Methods and kits for determining DNA repair capacity

ABSTRACT

In one aspect, the disclosure provides methods, compositions, vectors and kits for determining DNA repair capacity in cells or a subject.

RELATED APPLICATIONS

This application is a national stage filing under U.S.C. § 371 of PCTInternational Application PCT/US2012/056433, filed Sep. 20, 2012.Application PCT/US2012/056433 claims priority under 35 U.S.C. § 119(e)of U.S. provisional application Ser. No. 61/536,659, entitled “DNAREPAIR CAPACITY KIT” filed Sep. 20, 2011, the disclosure of which isincorporated by reference herein in its entirety.

GOVERNMENT SUPPORT

This invention was made with government support under Grant No. OD006422awarded by the National Institutes of Health. The government has certainrights in the invention.

FIELD OF THE INVENTION

The field of invention relates to methods, compositions, vectors andkits for determining DNA repair capacity.

BACKGROUND OF THE INVENTION

DNA is under constant assault from damaging agents that produce acomplex array of lesions. Left unrepaired, these lesions have thepotential to result in cell death, or may lead to mutations that alterthe biology of the cell and compromise the health of the organism,leading to degenerative diseases, cancer, and death. Consequently,inter-individual variation in the ability to respond to DNA damage is acritical component in answering a central question in biology: why dosome people develop disease, while others do not?

Numerous human diseases are associated with mutations in DNA repairgenes, and genetic tests are now available for a subset of highlypenetrant mutations. Oncologists have begun to use these tests to tailormedical treatment and prevention to individual needs, for example byrecommending specific cancer screening programs to patients based ontheir genetic susceptibility. Despite the enormous insight that has beengained from genetic screens, robust predictions of phenotype and diseasesusceptibility are hindered by epigenetic complexity, tissue-specificvariability in gene expression, uncharacterized mutations, andvariability in lifestyle and environmental exposure. Consequently,phenotypic screens are needed to complement genetic screens.

SUMMARY OF THE INVENTION

In one aspect, the disclosure provides methods, compositions, vectorsand kits for determining DNA repair capacity.

In one aspect, the disclosure provides methods of determining DNA repaircapacity in a cell. In some embodiments, the method of determining DNArepair capacity in a cell comprises introducing one or more DNA repairreporter vectors into a cell, and determining the capacity of the cellto process the one or more DNA repair reporter vectors therebydetermining the DNA repair capacity in the cell.

In one aspect, the disclosure provides methods of determining DNA repaircapacity of a subject. In some embodiments, the method of determiningDNA repair capacity of a subject comprises introducing one or more DNArepair reporter vectors into cells obtained from a subject, anddetermining the capacity of the cells to process the one or more DNArepair reporter vectors thereby determining the DNA repair capacity ofthe subject. In some embodiments, the cells obtained from the subjectare blood cells.

In one aspect, the disclosure provides methods of determining thepropensity of a subject to respond to a cancer treatment regimen. Insome embodiments, the method determining the propensity of a subject torespond to a cancer treatment regimen comprises introducing one or moreDNA repair reporter vectors into cells obtained from a subject, whereinthe one or more DNA repair reporter vectors comprise one or more lesionsthat are representative of a cancer treatment regimen, and determiningthe capacity of the cells to process the one or more DNA repair reportervectors thereby determining the propensity of the subject to respond tothe cancer treatment regimen. In some embodiments, the cells obtainedfrom the subject are cancer cells. In some embodiments, the methodfurther comprises comparing the capacity of the cancer cells to processthe one or more DNA repair reporter vectors to the capacity ofnon-cancer cells to process the one or more DNA repair reporter vectors.In some embodiments, the lesions that are representative of a cancertreatment regimen comprise DNA-crosslinks. In some embodiments, thelesions that are representative of a cancer treatment regimen compriseDNA lesions that block transcription. In some embodiments, the lesionsthat are representative of a cancer treatment regimen comprise DNAlesions that induce transcription errors. In some embodiments, thelesions that are representative of a cancer treatment regimen compriseDNA alkylation damage. In some embodiments, the lesions that arerepresentative of a cancer treatment regimen comprise O⁶-methyl-guanine.In some embodiments, the lesions that are representative of a cancertreatment regimen comprise N⁷-methylguanine.

In one aspect, the disclosure provides methods of determining thesusceptibility of a subject to an environmental condition. In someembodiments, the method of determining the susceptibility of a subjectto an environmental condition comprises introducing one or more DNArepair reporter vectors into cells obtained from a subject, wherein theone or more DNA repair reporter vectors comprise lesions that arerepresentative of an environmental condition, and determining thecapacity of the cells to process the one or more DNA repair reportervectors thereby determining the susceptibility of the subject to theenvironmental condition. In some embodiments, the environmentalcondition is sunlight exposure. In some embodiments, the lesions thatare representative of sunlight exposure comprise thymine dimers. In someembodiments, the environmental condition is ionizing radiation. In someembodiments, the lesions that are representative of ionizing radiationcomprise DNA double strand breaks. In some embodiments, theenvironmental condition is exposure to a carcinogenic compound. In someembodiments, the environmental condition is exposure to one of more ofthe conditions of Table A.

In some embodiments of any of the methods provided herein, processingthe one or more DNA repair reporter vectors comprises modifying a DNAlesion present in the one or more DNA repair reporter vectors.

In some embodiments of any of the methods provided herein, processing isdetected by a change in a fluorescence signal.

In some embodiments of any of the methods provided herein, processing isdetected by a change in the transcribed sequence of the one or more DNArepair reporter vectors.

In some embodiments of any of the methods provided herein, processing isdetected by a change in the amount of transcribed sequence of the one ormore DNA repair reporter vectors.

In some embodiments of any of the methods provided herein, DNA repair isnucleotide excision repair, homologous recombination, non-homologous endjoining, microhomology mediated end joining, direct reversal, baseexcision repair, mismatch repair or interstrand crosslink repair.

In one aspect, the disclosure provides methods of determining multipleDNA repair capacities in a cell. In some embodiments, the method themethod of determining multiple DNA repair capacities in a cell comprisesintroducing multiple DNA repair reporter vectors into a cell, anddetermining the capacity of the cell to process the multiple DNA repairreporter vectors thereby determining multiple DNA repair capacities inthe cell. In some embodiments, the multiple DNA repair reporter vectorscomprises at least two DNA repair reporter vectors. In some embodiments,the multiple DNA repair reporter vectors comprises at least four DNArepair reporter vectors. In some embodiments, each DNA repair reportervector of the multiple DNA repair reporter vectors comprises a uniqueDNA lesion. In some embodiments, each DNA repair reporter vector of themultiple DNA repair reporter vectors comprises a specific number of DNAlesions. In some embodiments, each DNA repair reporter vector of themultiple DNA repair reporter vectors comprises a number of DNA lesionscorresponding to a specific dose of damaging agent. In some embodiments,the multiple DNA repair reporter vectors comprise lesions susceptible toprocessing by nucleotide excision repair, homologous recombination,non-homologous end joining, microhomology mediated end joining, directreversal, base excision repair, mismatch repair or interstrand crosslinkrepair.

In one aspect, the disclosure provides kits comprising one or more DNArepair reporter vectors and instructions for use of the one or more DNArepair reporter vectors. In some embodiments, the kit comprises at leasttwo DNA repair reporter vectors. In some embodiments, the kit comprisesat least four DNA repair reporter vectors. In some embodiments, each DNArepair reporter vector of the kit comprises a unique DNA lesion. In someembodiments, the DNA repair reporter vectors of the kit comprise lesionssusceptible to processing by nucleotide excision repair, homologousrecombination, non-homologous end joining, microhomology mediated endjoining, direct reversal, base excision repair, mismatch repair orinterstrand crosslink repair. In some embodiments, the kit furthercomprises a cell line with a known DNA repair capacity.

In one aspect, the disclosure provides kits for determining thepropensity of a subject to respond to a cancer treatment regimencomprising one or more DNA repair reporter vectors, wherein the one ormore DNA repair reporter vectors comprise lesions representative of acancer treatment regimen, and instructions for use of the one or moreDNA repair reporter vectors. In some embodiments, the kit comprises atleast two DNA repair reporter vectors. In some embodiments, the kitcomprises at least four DNA repair reporter vectors. In someembodiments, each DNA repair reporter vector of the kit comprises aunique DNA lesion. In some embodiments, the kit further comprises a cellline with a known DNA repair capacity.

In one aspect, the disclosure provides kits for determining thesusceptibility of a subject to an environmental condition comprising oneor more DNA repair reporter vectors, wherein the one or more DNA repairreporter vectors comprise lesions representative of an environmentalcondition, and instructions for use of the one or more DNA repairreporter vectors. In some embodiments, the kit comprises at least twoDNA repair reporter vectors. In some embodiments, the kit comprises atleast four DNA repair reporter vectors. In some embodiments, each DNArepair reporter vector of the kit comprises a unique DNA lesion. In someembodiments, the kit further comprises a cell line with a known DNArepair capacity.

In one aspect, the disclosure provides kits for determining the repaircapacity of a cell line comprising one or more DNA repair reportervectors, wherein the one or more DNA repair reporter vectors allow forthe determination of the repair capacity of the cell line, andinstructions for use of the one or more DNA repair reporter vectors. Insome embodiments, the kit comprises at least two DNA repair reportervectors. In some embodiments, the kit comprises at least four DNA repairreporter vectors. In some embodiments, each DNA repair reporter vectorof the kit comprises a unique DNA lesion. In some embodiments, the kitfurther comprises a cell line with a known DNA repair capacity.

In one aspect, the disclosure provides a DNA repair reporter vectorcomprising a DNA lesion and a fluorescence reporter gene. In one aspect,the disclosure provides a DNA lesion and a first nucleic acid sequenceallowing for the identification of the DNA lesion. In one aspect, thedisclosure provides a DNA repair reporter vector comprising a DNA lesionand a first nucleic acid sequence allowing for the identification of theDNA lesion further comprising a fluorescence reporter gene. In someembodiments, the DNA repair reporter vector further comprises a secondnucleic acid sequence allowing for the identification of the DNA repairreporter vector. In some embodiments, the first nucleic acid sequenceallows for the determination of the processing of the DNA lesion. Insome embodiments, the processing is detected by a change in thetranscribed sequence of the nucleic acid sequence. In some embodiments,the processing is detected by a change in the amount of transcribedsequence of the nucleic acid sequence. In some embodiments, the DNAlesion is susceptible to processing by nucleotide excision repair,homologous recombination, non-homologous end joining, microhomologymediated end joining, direct reversal, base excision repair, mismatchrepair or interstrand crosslink repair. In some embodiments, the DNAlesion is representative of a cancer treatment regimen. In someembodiments, the DNA lesion is representative of an environmentalcondition.

These and other aspects and embodiments of the invention are describedin greater detail below.

Each of the limitations of the invention can encompass variousembodiments of the invention. It is, therefore, anticipated that each ofthe limitations of the invention involving any one element orcombinations of elements can be included in each aspect of theinvention. This invention is not limited in its application to thedetails of construction and the arrangement of components set forth inthe following description or illustrated in the drawings. The inventionis capable of other embodiments and of being practiced or of beingcarried out in various ways. Also, the phraseology and terminology usedherein is for the purpose of description and should not be regarded aslimiting. The use of “including”, “comprising”, or “having”,“containing”, “involving”, and variations thereof herein, is meant toencompass the items listed thereafter and equivalents thereof as well asadditional items.

BRIEF DESCRIPTION OF THE DRAWINGS

The figures are illustrative only and are not required for enablement ofthe invention disclosed herein.

FIG. 1 is an illustration showing an overview of the HT-HCR methodology.DNA lesions are introduced into fluorescent reporter plasmids in vitro.Numbers labeling the plasmids represent the dose (in J/m²) of UVradiation. Following treatment, plasmids were combined andco-transfected into cells. After 18 or 40 hours incubation, cells wereassayed for fluorescence by flow cytometry. Comparison of fluorescencesignals to those from cells transfected with undamaged plasmids yields adose-response curve (experimental data for GM02344 with plasmidcombination #1 in Table 2).

FIG. 2 shows the validation of HT-HCR against literature data. a)Dose-response curves for seven cell lines 18 hours after transfectionwith plasmid combination #1 (Table 2). Error bars represent the standarddeviation calculated from biological triplicates. b) Dose responsecurves at 40 hours. c) Comparison of % reporter expression as measuredby HT-HCR at 400 J/m² is plotted against % CAT as measured byconventional HCR for the same cell lines at 300 J/m². d) D_(o) valuescalculated from HT-HCR data plotted against those reported in theliterature.

FIG. 3 shows the reproducibility of the HT-HCR. a) Dose-response curvesfor seven cell lines 18 hours after transfection with plasmidcombination #2 (Table 2). b) Dose response curves at 18 hours. Errorbars represent the standard deviation calculated from biologicaltriplicates. c) Dose response curves at 40 hours. c) Comparison of %reporter expression as measured by HT-HCR at 400 J/m² is plotted against% CAT as measured by conventional HCR for the same cell lines at 300J/m². d) Comparison of HT-HCR data for plasmids treated at 400 J/m² inexperiments #1 and #2.

FIG. 4 shows measurements of DNA repair capacity in two cell types fromeach of seven individuals. a) Dose response curves generated by HT-HCRfor lymphoblastoid cell lines 40 hours after transfection with plasmidcombination #2 (Table 2). Error bars represent the standard deviationcalculated from biological triplicates. b) Corresponding dose responsecurves for primary skin fibroblasts from the same seven individuals. c)Correlation between % reporter expression in the two cell types at 800J/m².

FIG. 5 shows the construction and validation of a plasmid containing asite-specific thymine dimer. a) A synthetic oligonucleotide bearing asite-specific thymine dimer is ligated into a gapped plasmid accordingto the methods of Kitsera et al. (a). b) Gel electrophoresis ofsynthetic intermediates and product (lanes 1-4) and assay for sitespecific or randomly introduced pyrimidine dimers in reporter plasmids(lanes 5-6). c) NER-dependent reduction in fluorescent reporterexpression from plasmids containing the site-specific thymine dimer.

FIG. 6 shows the validation of HCR-Seq against HT-HCR. a) workflow forexperiments comparing three methods of quantifying reporter expression.b) Dose response curves generated for cells transfected with plasmidcombination #3 from Table 2, and assayed by HT-HCR. Error bars representthe standard deviation of biological duplicates. c) Correspondingdose-response curves generated from RNAseq data. In the pie chart, thefraction of mapped reads aligning to reporter transcripts is representedin black, and all other reads are represented in gray. d) Dose responsecurves generated from DNAseq data. Reads aligning to reporters are againrepresented in black, and all others are represented in gray.

FIG. 7 shows transcriptional mutagenesis opposite a site-specificthymine dimer. a) Percentage of reads containing a deletion that spansthe site of the thymine dimer, as measured by RNAseq (the sequences,from top to bottom, correspond to SEQ ID NOs.: 8 and 9) or b) DNAseq(the sequence corresponds to SEQ ID NO: 8). c) Percentage of reads inwhich guanine has been misincorporated in place of the expected 3′adenine in the sequence opposite the thymine dimer, measured by RNAseqor d) DNAseq.

FIG. 8 shows chromatin immunoprecipitation from three types of cellstransfected with pmax:mCherry A) lymphoblasts, B) fibroblasts, C) mousesplenic lymphocytes.

FIG. 9 shows gel electrophoresis of amplicons generated from reportercDNAs by PCR.

FIG. 10 shows genes expression profile of cells transfected with damagedor undamaged reporter plasmids. In panels A and B, levels of expressionare plotted for all transcripts. Levels of expression in cells that weretransfected with damaged plasmids are plotted on the vertical axis, andexpression levels in cells transfected with the undamaged (control)plasmids are plotted on the horizontal. Genes expressed at the samelevel under both conditions appear on the diagonal, and this isoverwhelmingly the case for endogenous transcripts (black and graycircles), indicating no major changes in transcription in cells inresponse to the presence of damaged plasmid DNA. Reporter transcriptsare colored in blue, cyan, orange, green, and magenta. These reportersare seen to be among the most highly expressed in all samples. Reducedexpression in the presence of DNA damage (due to transcription blockinglesions) is reflected in these points falling below the diagonal

FIG. 11 shows expression of GFP from reporter plasmid containing a sitespecific thymine dimer in the transcribed strand, assayed by flowcytometry, RNAseq, or DNAseq. Error bars represent the standarddeviation of two biological replicates

FIG. 12 shows read coverage and junction reads for GM02344 “XPA^(mut)”and GM01953 “XPA^(WT)”. Reads are aligned to the region of the genomethat encodes the XPA gene (diagrammed at the bottom of the figure). Themajority of reads in GM01953 align, as expected, to the exons (3, 4, and5 are shown). Read coverage is overall higher and the expectedintron-spanning reads (indicated as light blue lines that run betweenexons) are abundant. By contrast, read coverage is lower, intronspanning reads are nearly absent, and a significant number of readswithin the introns all support an elevated frequency of splicing errorsin the XPA gene in GM02344.

FIG. 13 shows the frequency of deletions of 3 or more base pairs andspanning ApA sites in reads aligning to randomly damaged reportersequences. Dark grey bars represent GM01953 (WT) and light greyrepresents GM02344 (XPA mutant). In the absence of UV irradiation, abackground frequency of about 10 per million reads is observed. Thefrequency rises to about 50 reads per million in GM01953, and stillhigher to about 100 reads per million in GM02344. This pattern issimilar to that observed for the frequency of deletions at the positionof the site specific lesion, providing additional evidence that thedeletions are due to error-prone transcriptional bypass of unrepairedbulky DNA adducts.

FIG. 14 is a drawing showing embodiments of kits for DNA repair capacitymeasurements. One kit uses reporters with fluorescence based detection(Lumens) and one kit uses next generation sequencing based detection(Sequens).

FIG. 15 is a sequence (SEQ ID NO:3) showing the construction of reporterconstructs.

FIG. 16 is a drawing illustrating the multicolor fluorescent reporterstrategy. The column graphs show expected data for reporter expressionfrom DNA repair reporter vectors irradiated with UVC at several doses.

FIG. 17 is a drawing that shows flow cytometric detection andmeasurement of reporter fluorescence. The wavelength of the laser usedto excite fluorophores is given in nm.

FIG. 18 is a graph of a dose response curve corresponding to themulticolor fluorescent reporter strategy shown in FIG. 16.

FIG. 19 is a graph showing the correlation between D_(o) as calculatedby Athas et al. and D_(o) obtained using the developed assay.

FIG. 20 is an illustration of sequencing based detection of reportertranscripts.

FIG. 21 is an illustration of the methodology for analysis of reportertranscripts by next generation sequencing. Transcripts can be analyzeddirectly by RNAseq or can be converted to cDNA and selectively amplifiedto increase the signal-to-noise ratio for DNA sequencing.

FIG. 22 is an image of a gel purification of cDNA amplified usingreporter-specific primers. The gel shows cDNA amplified with the 5′ and3′ UTR primers shown in FIG. 15.

FIG. 23 is an illustration of the utility of single nucleotideresolution transcript analysis. (SEQ ID NO:4 and SEQ ID NO:5). The datain the column graphs are expected data based on the relative abundanceof reporter transcripts for the lesion shown.

FIG. 24 is an illustration of a method used for the introduction ofsite-specific DNA lesions into reporter plasmids.

FIG. 25 is an illustration of the introduction of a site-specificthymine dimer into the pmax reporter plasmid.

FIG. 26 is an image with a corresponding graph illustrating theverification of a site-specific DNA damage containing reporter.

FIG. 27 is an illustration showing the finding that four basic reporterconstructions comprise the library of reporters to be used with nextgeneration sequencing. A-D represent DNA sequences in the context of atransiently transfected plasmid reporter.

FIG. 28 shows a primary gating scheme for TK6 lymphoblastoid cells.

FIG. 29 shows a gating scheme for negative controls (mock transfectedcells).

FIG. 30 provides an example of a single color control gating, wheresingle color refers to cells transfected with a single fluorescentreporter plasmid.

FIG. 31 shows Cyan Excluded from transfection. The number of cellsappearing in both P13 and P14 is zero, consistent with the absence ofthe AmCyan reporter in the transfection. All other reporters aredetected.

FIG. 32 is a graph showing the dose-dependent inactivation offluorescent DNA repair reporter plasmids treated with Cisplatin.

FIG. 33 is an illustration showing the synthesis of a substrate with asite-specific O⁶-MeG.

FIG. 34 shows graphs of a DNA repair capacity assay using a fluorescentreporter vector with a site-specific O⁶-MeG.

FIG. 35 is a graph comparing 2-color versus 5-color fluorescent reporterHCR of UV-irradiated plasmids. UV HCR: XPA-deficient cell line at 16hours. The number of colors refers to the number of fluorescentreporters co-transfected into cells. The color of the fluorescentreporter used at each dose in the 5-color experiment is indicated by anarrow. In the 2-color experiment, mCherry was used at all 4 doses inseparate transfections.

FIG. 36 is an illustration showing the estimation of recombinationfrequency.

FIG. 37 is a graph showing a 25-fold range of HR repair capacity overseveral cell lines in DSB (double strand break) “induced” recombination.

FIG. 38 is an illustration showing the mismatch repair substrate withmethods adapted from Zhou, B. S. et al, Anal. Biochem. 388, 167-169,(2009).

FIG. 39 is an illustration showing multiple lesions in a single plasmid.A single base loop is indicated at position 50, an A:C mismatch is shownat position 215, and a G:G mismatch is shown at position 299. All threelesions are substrates for mismatch repair.

FIG. 40 is a graph showing that the ability to distinguish between therepair capacity of MMR+ and MMR− cells improves with multiple lesions.

FIG. 41 is a graph showing that the inhibition of transcription is notdetected when a reporter plasmid is treated with MNNG, followed bytransfection into cells, and a fluorescence assay is performed at 16hours. In this example a plasmid was treated for 4 hours in 0.8 mM MNNG.

FIG. 42 shows that a point mutation (T208C) results in a non-fluorescentmPlum variant S70P. Of 500,000 cells analyzed—only a single plumpositive cell was found.

FIG. 43 is an illustration showing that when O⁶MeG is present in thetranscribed strand, some mRNA will contain U, and will be translatedinto wild type mPlum protein.

FIG. 44 is an illustration showing an assay for O⁶Methylguanine HCR.

FIG. 45 shows the results for TK6, MGMT-deficient (MGMT=methyl guaninemethyl transferase). 500,000 cells were analyzed—a few hundred plumpositive TK6 cells are detected following transfection with MNNG treatedplasmid, consistent with alkylation induced transcriptional mutagenesis.

FIG. 46 is a bar graph showing that the lack of signal is MGMTdependent. BnG stands for O⁶-benzylguanine, which inhibits MGMT.

FIG. 47 shows a preliminary comparison with independent characterizationof MGMT activity in extracts. (Fry et al Genes. Dev. 2008 (22) p 2621).

FIG. 48 shows that measurement of NER and HR in a single assay yieldsthe same information as separate measurements. “Separate” refers to anexperiment in which plasmid reporters for NER and HR capacity weretransfected separately. “Together” refers to an experiment in which thereporters for NER and HR capacity were co-transfected in a single assay.

FIG. 49 shows HCR of plasmids containing etheno (ϵ) lesions. The upperleft panel shows mass spectrometric quantitation of etheno adducts inplasmid DNA treated with chloroacetaldehyde (CAA). Dose and cell linedependent inhibition of transcription in plasmids treated with CAA isshown at right.

FIG. 50 shows that mouse cells deficient for base excision repair anddirect reversal of (ϵ) lesions (Aag, Alkbh2, Alkbh3 null) exhibitreduced expression of fluorescent reporters that have been damaged withCAA.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect, the disclosure provides methods, vectors, compositionsand kits for determining DNA repair capacity in a cell or a subject.

In one aspect, the disclosure provides methods of determining DNA repaircapacity in a cell. In some embodiments, the disclosure provides methodsof determining DNA repair capacity in a cell, the method comprisingintroducing one or more DNA repair vectors into a cell, and determiningthe capacity of the cell to process the one or more DNA repair vectorsthereby determining the DNA repair capacity of the cell.

DNA is constantly bombarded by both endogenous and environmental DNAdamaging agents. Failure to repair this damage is a risk factor fordisease, and the available data point to a wide range of capacity torepair DNA damage in the human population. However, prior to methodsprovided in the instant disclosure, the complexity and multitude of DNArepair pathways have thus far precluded large-scale comprehensiveinvestigations of inter-individual differences in DNA repair capacity.

In one aspect, the disclosure provides multiplexed, high-throughput,quantitative assays for DNA repair capacity. In some embodiments, theDNA repair capacity is evaluated through fluorescence reporters. In someembodiments, a high throughput flow cytometric host cell reactivationassay (HT-HCR) with six fluorescent reporters is provided (Also referredto herein as “Lumens”). Unexpectedly, it was found that a system withthe multiple reporters could be used to evaluate one or more aspects ofthe DNA repair capacity in a cell or subject. In some embodiments, theHT-HCR assay has been used to measure simultaneously host cellreactivation of multiple plasmid DNA reporters treated with severaldoses of UVC.

In one aspect, the disclosure provides sequencing based methods todetermine the DNA repair capacity in a cell. In some embodiments, thesequence based method is a next generation sequencing based host cellreactivation assay (HCR-Seq; Also referred to herein as “Sequens”). Itwas surprisingly found that sequencing based methods allow for themultiplex detection of the DNA repair of any DNA repair pathway. Asprovided herein, in some embodiments, in a single DNA sequencing lane 40reporters or more could be detected simultaneously and independently.Thus, for the first time a method is provided that allows for theindependent detection of multiple reporters (up to 4000, or more),allowing for studies of DNA repair capacity in large populations. Thesingle nucleotide resolution of the HCR-Seq data permits discovery andquantitation of rare transcriptional mutagenesis events due to lesionbypass by RNA polymerase, and thus allows for the detection of DNArepair of any DNA repair pathway acting on a lesion that changes thetranscribed sequence or the amount of transcript produced. Such lesionsexist for all of the major DNA repair pathways. The methods providedherein allow for a synthesis of gene expression patterns and proteinbiomarkers that can help to personalize the prevention, diagnosis andtreatment of cancer in a single assay. The hurdles of molecularcomplexity and heterogeneity among individuals and across disease statescan be addressed by determining the DNA repair capacity according to themethods provided herein.

Direct evidence of a relationship between DNA repair capacity and cancersusceptibility comes from several studies of nucleotide excision repair(NER) [5]. Individuals with Xeroderma Pigmentosum (XP) are highlydeficient for NER, and suffer an estimated 2000-fold increased risk ofdeveloping cancer in sun-exposed skin [6]. Epidemiological studies inapparently healthy individuals demonstrated a wide range of NERcapacity, and an apparent link between NER capacity and cancersusceptibility [5, 7-9]. Additional studies have examined links betweenother repair pathways and cancer [10, 11].

Despite a clear relationship between DNA repair capacity and humanhealth, the majority of these epidemiological studies have beenrelatively small, and most have been focused on the NER pathway. A majorbarrier to larger studies that encompass additional pathways is the lackof a high throughput assay for DNA repair capacity in multiple DNArepair pathways.

In one aspect, the disclosure provided host cell reactivation (HCR)assay. Host cell reactivation (HCR) assays allow for the measuring DNArepair capacity in intact cells by using DNA repair reporter vectors. Insome embodiments, in a typical HCR assay, a plasmid that expresses areporter gene is exposed to a DNA damaging agent that generatestranscription-blocking lesions, and then the plasmid is transfected intoliving cells. In the absence of repair, expression of the reporter geneis inhibited. However, if the cells are able to repair the damage, thetranscription blocking lesions are removed, and expression of thereporter is reactivated.

Prior to the instant disclosure a high throughput assay system fordetermining DNA repair capacity was not available. Up to now, thestandard reporter used to determine DNA repair capacity ischloroamphenicol acetyl transferase (CAT). A major limitation of thisreporter is the need to lyse cells following transfection withsubsequent liquid scintillation or thin layer chromatography analysis,and the requirement of radiolabeled substrates. A further limitation isthe inability to control for transfection efficiency. The need forradiolabels and the inability to control for transfection efficiencyhave been overcome through the use of a dual luciferase reporter [16],however this methodology retains the need to lyse cells and depends onexogenous reagents that must be added to lysates to assay for thereporter. While fluorescent reporters have become available [17], it hasnot been possible to multiplex such assays. Prior to the methodsprovided herein multiplex determination of DNA repair capacity could notbe achieved because of (i) the inability to measure more than one DNArepair signal at a time, (ii) the lack of a general method of detectingrepair of lesions that can be bypassed by RNA polymerase and (iii) thelack of a thorough validation of fluorescent reporter systems againstthe standard CAT reporter systems.

In one aspect, the disclosure provides multiplex methods for determiningDNA repair capacity in a cell. In some embodiments, the multiplex assayis a fluorescence based assay. In some embodiments, the multiplex assayis a high throughput host cell reactivation assay (HT-HCR) that makesuse of multiple fluorescent reporter proteins in a single assay tomeasure multiple separate repair processes. In some embodiments, themultiplex assay is a sequence based assay. In some embodiments, themultiplex assay is HCR-Seq, a method that provides the ability tomeasure thousands of independent repair processes in a single assay.

Determining DNA Repair Capacity

The genetic material of cells is continually challenged by intracellularand extracellular conditions, such as cell metabolites, radiation andexposure to environmental agents. Exposure to these conditions can leadto DNA damage. DNA damage is unwanted because it can lead to theblocking of replication, which would result in the death of the cell,blocking of transcription which may lead to the cell death if essentialproteins are involved, and mutagenesis. Mutagenesis may be lethal if thefunction of an essential protein is compromised or, can result inuncontrolled cell growth (e.g. carcinogenesis) if the activity of atumor suppressor gene is compromised. Cells have evolved a variety ofDNA repair mechanisms to remove or suppress DNA damage. These mechanismsinclude nucleotide excision repair, which is generally directed againstmulti base pair lesions, base excision repair, which is generallydirected against single base lesion, mismatch repair, which removesmismatched base pairs, end joining, which is directed against doublestrand breaks, direct reversal, which generally removes chemicalmodifications from DNA bases, and recombinational repair, which removesa variety of lesions including deletions and replication blockinglesions (An overview of the various DNA repair pathways can be found forinstance in DNA Repair and Mutagenesis, Friedberg et al., ASM Press).Each repair pathway uses a variety of DNA repair enzymes that act on andprocess the DNA lesion. The importance of DNA repair is exemplified inpeople who are deficient for a particular repair mechanism. Forinstance, subjects suffering from Xeroderma Pigmentosum are deficient inone of the proteins of the nucleotide excision repair pathway. Thesesubjects have only a very limited ability to process cyclobutanepyrimidine dimer DNA lesions (such as thymine dimers) and (6-4)photoproducts that are caused by exposure to sunlight, and they are athigh risk for developing skin cancers. Similarly, people with mutationsin genes coding for enzymes involved in mismatch repair have been shownto be at increased risk for colorectal cancer.

In one aspect, the disclosure provides a method of determining the DNArepair capacity of a subject. In some embodiments, the method ofdetermining the DNA repair capacity of a subject comprises introducingone or more DNA repair reporter vectors into cells obtained from asubject, and determining the capacity of the cells to process the one ormore DNA repair reporter vectors thereby determining the DNA repaircapacity of the subject. In some embodiments, the cells obtained fromthe subject are blood cells.

In one aspect, the disclosure provides a method of determining thepropensity of a subject to respond to a cancer treatment regimen. Insome embodiments, the method of determining the propensity of a subjectto respond to a cancer treatment regimen comprises introducing one ormore DNA repair reporter vectors into cells obtained from a subject,wherein the one or more DNA repair reporter vectors comprise one or morelesions that are representative of a cancer treatment regimen, anddetermining the capacity of the cells to process the one or more DNArepair reporter vectors thereby determining the propensity of thesubject to respond to the cancer treatment regimen. In some embodiments,the cells obtained from the subject are cancer cells. In someembodiments, the method of determining the propensity of a subject torespond to a cancer treatment regimen further comprises comparing thecapacity of the cancer cells to process the one or more DNA repairreporter vectors to the capacity of non-cancer cells to process the oneor more DNA repair reporter vectors.

Every person has a unique DNA repair capacity profile which iscorrelated to the activity of specific DNA repair pathways (See e.g.,Fry, R. C. et al. Genomic predictors of inter-individual differences inresponse to DNA damaging agents. Genes Dev. 22, 2621-262). Thus, forinstance, a first person may have a very active nucleotide excisionrepair but may have low base excision repair activity. A second personmay have both low activity for nucleotide excision repair and baseexcision repair but may have a high recombinational repair activity.Some people may have more activity in all DNA repair pathways comparedto other people. When a person is exposed to a DNA damaging event, suchas cancer chemotherapy, sunlight or a different environmental condition,resulting in DNA lesions, the response to these lesions will vary fromperson to person depending on their DNA repair capacity profile. Thedisclosure provides methods to assess how a person will respond to avariety of DNA damaging events.

In one aspect, the disclosure provides methods of determining DNA repaircapacity in a cell. In some embodiments, the method of determining DNArepair capacity in a cell comprises introducing one or more DNA repairreporter vectors into a cell, and determining the capacity of the cellto process the one or more DNA repair reporter vectors therebydetermining the DNA repair capacity in the cell. The methods of thedisclosure are not limited to a particular cell type and any cell intowhich DNA repair vectors can be introduced can be evaluated according tothe methods of the invention. Cells of interest, for instance, are cellsof which the DNA repair capacity is unknown or cells that are to bechallenged with one or more conditions that can cause DNA damage. Cellsin which the DNA repair capacity is to be determined can be cellsobtained from subjects, including humans and animals. Cells obtainedfrom a subject include blood cells, skin cells and cancerous cells(e.g., through biopsy). Cells in which the DNA repair capacity is to bedetermined can be transformed cells that can divide indefinitely, orprimary cells that are expected to divide only a limited number oftimes. In some embodiments, the cells in which the DNA repair capacityis to be determined are cancer cells. Determining the DNA repaircapacity in a cancer cell provides insight into therapeutic treatmentoptions for the particular cancer regimen. Thus, established cancer celllines can be assayed for their repair capacities. For instance, a coloncancer cell line can be investigated for its proficiency in baseexcision repair and nucleotide excision repair. If the cancer cell lineshows to be more proficient in base excision repair than in nucleotideexcision repair the cancer could be treated with a chemotherapeuticagent (e.g., cisplatin), which results in DNA damage which is normallyprocessed by nucleotide excision repair. Because the cancer is lessproficient in repairing lesions processed by nucleotide excision repair,subjecting the cell to such lesions should offer a preferred way ofkilling the cell. Knowing the repair capacity of the cancer cell line inmultiple DNA repair pathways helps to refine the chemotherapeuticwindow. If the cancer cell line is deficient in direct reversal ofO⁶-methylguanine lesions, but is proficient in all other pathways, thecancer could be treated with a chemotherapeutic agent (e.g.,temozolomide) that induces O⁶-methylguanine. If however the cancer cellline is deficient in both direct reversal of O⁶-methylguanine andmismatch repair, the cells will not be sensitive to chemotherapeuticagents that induce O⁶-methylguanine, and a different route of treatmentshould be sought. In some embodiments, the DNA repair profile of acancer obtained from a subject can be evaluated prior tochemotherapeutic treatment. Thus, a cancer cell can be obtained from asubject and the DNA repair profile of the cancer cell evaluated. Thesubsequent chemotherapy can take into account the DNA repair profile ofthe cancer cell. In some embodiments, the DNA repair profile of thecancer cell of a subject can be compared to the DNA repair profile ofnon-cancerous cells from the subject. Thus, a therapeutic window can bedeveloped by comparing the DNA repair profile of a cancer cell of asubject to the DNA repair of a non-cancerous cell in subject. Thismethod of maximizing the efficacy of treatment while minimizing sideeffects to the patient avoids the still common practice of determiningthe best chemotherapy regimen by trial-and-error.

In some embodiments, one or more cell types can be obtained from asubject and the DNA repair profile of the cells determined. In someembodiments, the DNA repair profile of subject is determined bydetermining the DNA repair profile of one or more cell types obtainedfrom the subject. In some embodiments, the DNA repair profile of asubject can be determined by determining the DNA repair profile of arepresentative cell type. In some embodiments, the representative celltype is a blood cell. Knowing the DNA repair profile of subject allowsfor the development of the therapeutic window and of the tailoring ofcancer chemotherapy. Thus, a person classified as being highlyproficient in DNA base excision repair can be given an appropriate dose(e.g., increased dose) of cancer chemotherapy that induces DNA lesionsthat are processed by base excision repair. The healthy cells of asubject as being highly proficient in DNA base excision repair areexpected to process the increased number of DNA lesions caused by thechemotherapeutic agent because of the increased proficiency in baseexcision repair.

In some embodiments, the DNA repair profile of a person is compared tothe DNA repair profile of a cancer cell line. For instance, the DNArepair profile of a particular cancer cell may be known from previousexperiments. For instance, it may be known that melanoma is particularlysusceptible to ionizing radiation in general, and the susceptibility ofthe cancer cell may be correlated against the DNA repair profile of theperson, e.g., as determined by evaluating the DNA repair profile of arepresentative cell line of that person. An appropriate chemotherapeuticregimen may be decided on based on the DNA repair capacity of thesubject and the DNA repair profile of the cancer cell.

In one aspect, the disclosure provides methods of determining thesusceptibility of a subject to an environmental condition. In someembodiments, the method of determining the susceptibility of a subjectto an environmental condition comprises introducing one or more DNArepair reporter vectors into cells obtained from a subject, wherein theone or more DNA repair reporter vectors comprise lesions that arerepresentative of an environmental condition, and determining thecapacity of the cells to process the one or more DNA repair reportervectors thereby determining the susceptibility of the subject to theenvironmental condition. In some embodiments, the environmentalcondition is sunlight exposure. In some embodiments, the lesions thatare representative of sunlight exposure include thymine dimers. In someembodiments, the environmental condition is ionizing radiation. In someembodiments, the lesions that are representative of ionizing radiationinclude DNA double strand breaks. In some embodiments, the environmentalcondition is exposure to a carcinogenic compound.

In one aspect, the disclosure provides methods of determining thesusceptibility of a subject to an environmental condition. Cells andsubjects are constantly challenged by conditions that can damage DNA.Exposure to some of these conditions cannot be avoided, e.g., oxidativedamage from cell metabolism. However, exposure to some of theseconditions that can damage DNA can be avoided or minimized (e.g.,ionizing radiation, sunlight). In one aspect, the disclosure providesmethods for providing subjects with information allowing the subjects tomodify their behavior accordingly. For instance, the methods providedherein allow for the determination of a subject's susceptibility tosunlight. A representative cell from a subject (e.g. skin cell or bloodcell) can be investigated for its ability to process DNA damage causedby sunlight. If a person has only a low ability to process DNA lesionscaused by sunlight that person should avoid sunlight in order tominimize the chance of developing skin cancer. If a subject has cellsthat are highly proficient in repairing DNA lesions caused by sunlightthat person does not need to be extremely diligent in avoiding sunlight.According to the methods provided herein subjects can be evaluated fortheir susceptibility to a variety of environmental conditions. Forinstance, a subject can be investigated for its susceptibility toionizing radiation, an environmental carcinogen, etc. Once a subjectknows its susceptibility to a particular environmental condition, thatperson can alter its lifestyle and try to minimize exposure to theenvironmental condition.

Lesions

In one aspect, the disclosure provides methods, compositions, vectorsand kits for determining the DNA repair capacity in a cell or a subject.In one aspect, the disclosure provides DNA repair reporter vectors fordetermining the DNA repair capacity in a cell or a subject. In someembodiments, the DNA repair reporter vectors include one or morelesions. A “lesion”, or “DNA lesion”, as used herein, refers to anystructural modification of the DNA that distinguishes the DNA fromcorrectly base-paired DNA, DNA lesion include DNA base modifications,mutations, deletions, DNA cross-links, uracil incorporation,modifications of the phosphodiester backbone etc. (See also Tables A-Dbelow). For instance, lesions include DNA alkylation lesions (See e.g.,Shrivastav et al., Carcinogenesis 31, 2010, p 59-70), oxidative DNAdamage (Cooke et al., FASEB J 17, 195-1214), and mismatches betweennatural and non-natural DNA base pairs.

In one aspect, the lesions used in the methods, compositions, vectorsand kits provided herein are representative of a cancer chemotherapyregimen. That is, the lesion is generated by exposing cells and/or asubject to cancer chemotherapy. For instance, alkylating agents such asnitrosoureas are commonly used in cancer chemotherapy. Exposure of cellsto nitrosourea results in a variety of DNA lesions includingO⁶-methyl-guanine. Thus, the lesion O⁶-methyl-guanine is representativeof a cancer chemotherapy regime using nitrosoureas, and such lesions canbe used according to the methods provided herein to determine thesusceptibility of a cell or subject to that particular cancerchemotherapy regimen. Similarly, an MNNG (N-methyl-N′-nitro-Nnitrosoguanidine) based cancer chemotherapy regimen would result inN⁷-methylguanine lesions in the DNA. DNA repair reporter vectorscomprising N⁷-methylguanine can therefore be used to determine thesusceptibility of a cell or subject to a cancer chemotherapy regimenthat includes MNNG. Analogously, cancer chemotherapy that includesionizing radiation will result in oxidative lesions and DNA strandbreaks and oxidative lesions and DNA strand breaks can are thereforerepresentative of cancer chemotherapy regimen that includes ionizingradiation. It should be appreciated that lesions can be representativefor more than one cancer chemotherapy regimen, and that more than onelesion may be necessary to fully represent a chemotherapy regimen. Forinstance, a DNA strand break can be generated by exposure to a varietyof lesions, and MNNG generates O⁶-methyl-guanine in addition toN⁷-methylguanine. A person of skill in the art can rely on theliterature to provide others lesions that representative of a particularcancer chemotherapy regimen.

In some embodiments of the methods, compositions, vectors and kitsprovided herein the lesions that are representative of a cancertreatment regimen comprise DNA-crosslinks. In some embodiments, thelesions that are representative of a cancer treatment regimen compriseDNA lesions that block transcription. In some embodiments, the lesionsthat are representative of a cancer treatment regimen comprise DNAlesions that induce transcription errors. In some embodiments thelesions that are representative of a cancer treatment regimen compriseDNA alkylation damage. In some embodiments, the DNA alkylation damagecomprises O⁶-methyl-guanine. In some embodiments, the DNA alkylationdamage comprises N⁷-methylguanine.

In one aspect, the lesions used in the methods, compositions, vectorsand kits provided herein are representative of environmental conditions.Similarly to the cancer treatment regimens discussed above, certainenvironmental conditions will result in specific DNA lesions. Forinstance, sunlight exposure will result in the formation of thyminedimers in cells, which are therefore representative of exposure tosunlight. It should be appreciated that lesions can be representativefor one or more environmental conditions and that more than one lesionmay be necessary to fully represent an environmental condition. Itshould also be appreciated that lesions can be representative for one ormore environmental conditions and one/or more cancer treatment regimens.In some embodiments, the environmental condition is sunlight exposure.In some embodiments, the lesion that is representative of sunlightexposure is a thymine dimer. In some embodiments, the environmentalcondition is ionizing radiation. In some embodiments, the lesion that isrepresentative of ionizing radiation is a DNA double strand breaks. Insome embodiments, the environmental condition is a carcinogeniccompound. Embodiments of additional environmental conditions andrepresentative DNA lesions are provided in Table A. In addition, aperson of ordinary skill in the art can rely on the literature toprovide information on additional lesions that are representative ofenvironmental conditions.

TABLE A Environmental conditions and representative DNA lesionsRepresentative Exposure damaging Route of A Representative Conditionagent exposure Types of Lesions Lesion References Sunlight UV light SkinCyclobutane thymine-thymine [1] pyrimidine cis-syn dimers and (6-4)cyclobutane photoproducts dimer Ionizing X-rays, free Ambient Strandbreaks, Double strand [2] Radiation radicals radon gas, X- free radicalbreak irradiation induced damage Tobacco Polycyclic InhalationBenzo[a]pyrene (+)-anti- [3] smoke aromatic adducts benzo[a]pyrenehydrocarbons diol epoxide -N²- dG Aspergillus Aflatoxin IngestionAflatoxin 8,9-dihydro-8- [4] (fungus) adducts (N7-guanyl)-9-hydroxyaflatoxin B₁ Drugs Psoralen Topical, DNA cross-links Psoralen [5]ingestion, (various drug Interstrand cross- injection dependent linklesions) Carcinogens Heterocyclic Ingestion amino- N-(deoxyguanosin- [6]in food Amines imidazoazaarene 8-yl)- 2-amino-3- adductsmethylimidazo[4,5- f]quinoline Air Reactive Inhalation Hydrogen 1-N⁶-[7] pollution oxygen, peroxide ethenoadenine nitrogen and free radicalsExhaust Polycyclic Inhalation Benzo[a]pyrene (+)-anti- [3] Fumesaromatic adducts benzo[a]pyrene hydrocarbons diol epoxide -N²- dGIndustrial N-2-Acetyl-2- Skin, eye, Aromatic amines N-(2′- [8] ChemicalsAminofluorene ingestion (various deoxyguanosin-8- chemicalyl)-N-acetyl-2- dependent aminofluorene lesions) Helicobacter ReactiveInfection Oxidative DNA 8-oxo-guanine [9] Pylori oxygen, damage nitrogenand free radicals Irritants in Reactive Inflammatory Oxidative andThymine glycol [10] food oxygen, response to free radical nitrogen andingestion (eg induced damage free radicals gluten in coeliacs) Burningof Methyl halides Inhalation Alkylation O⁶-methylguanine [11] biomassdamage of DNA bases 1. Setlow, R.B. and W.L. Carrier, Pyrimidine dimersin ultraviolet-irradiated DNAs. Journal of Molecular Biology, 1966.17(1): p. 237-&. 2. Iliakis, G., The role of DNA double strand breaks inionizing radiation-induced killing of eukaryotic cells Bioessays, 1991.13(12): p. 641-648. 3. Perlow, R.A., et al., DNA adducts from atumorigenic metabolite of benzo a pyrene block human RNA polymerase IIelongation in a sequence- and stereochemistry-dependent manner. Journalof Molecular Biology, 2002. 321(1): p. 29-47. 4. Essigmann, J.M., etal., Structural identification of major DNA adduct formed byaflatoxin-B1 in vitro. Proceedings of the National Academy of Sciencesof the United States of America, 1977. 74(5): p. 1870-1874. 5. Cole,R.S., Psoralen monoadducts and interstrand cross-links in DNA.Biochimica Et Biophysica Acta, 1971. 254(1): p. 30-&. 6. Schut, H.A.J.and E.G. Snyderwine, DNA adducts of heterocyclic amine food mutagens:implications for mutagenesis and carcinogenesis. Carcinogenesis, 1999.20(3): p. 353-368. 7. Marnett, L.J., Oxyradicals and DNA damage.Carcinogenesis, 2000. 21(3): p. 361-370. 8. Westra, J.G., E. Kriek, andH. Hittenhausen, Identification of persistently bound form of carcinogenN-acetyl-2-aminofluorene to rat liver DNA in vivo Chemico-BiologicalInteractions, 1976. 15(2): p. 149-164. 9. Touati, E., et al., Deficiencyin OGG1 protects against inflammation and mutagenic effects associatedwith H-Pylori infection in mouse. Helicobacter, 2006. 11(5): p. 494-505.10. Svilar, D., et al., Base Excision Repair and Lesion-DependentSubpathways for Repair of Oxidative DNA Damage. Antioxidants & RedoxSignaling, 2011. 14(12): p. 2491-2507. 11. Bolt, H.M. and B. Gansewendt,MECHANISMS OF CARCINOGENICITY OF METHYL HALIDES. Critical Reviews inToxicology, 1993. 23(3): p. 237-253.

In one aspect, the disclosure provides methods of determining the DNArepair capacity in a cell or subject. In order to determine the DNArepair capacity in a cell or subject it may be desirable to determinethe proficiency of one or more DNA repair pathways. In some embodiments,the disclosure provides methods allowing the determination of theproficiency of one or more DNA repair pathways in a cell or subject.Prior to the methods provided herein, it was not possible to adequatelydetermine the proficiency of more than one DNA repair pathway in a cellusing a single assay. In some embodiments, the disclosure providesmethods using lesions that are susceptible to processing by one more DNArepair mechanisms to evaluate the DNA repair capacity. In someembodiments, the lesions are susceptible to processing by nucleotideexcision repair, homologous recombination, non-homologous end joining,microhomology mediated end joining, direct reversal, base excisionrepair, mismatch repair or interstrand crosslink repair. In one aspect,the disclosure provides DNA repair reporter vectors comprising lesionsthat are susceptible to processing by one more DNA repair mechanismsthat are used to evaluate the DNA repair capacity. In some embodiments,the lesions are susceptible to processing by nucleotide excision repair,homologous recombination, non-homologous end joining, microhomologymediated end joining, direct reversal, base excision repair, mismatchrepair or interstrand crosslink repair.

In one aspect, the lesions used in the methods, compositions, vectorsand kits provided herein are susceptible to processing by a specific DNArepair pathway. Thus, lesions can be parsed with a particular DNA repairpathway that acts on the lesion. Representative lesions that can be usedto evaluate the proficiency of a DNA repair pathway are provided inTable B. For instance, nucleotide excision repair acts on thymine dimersand a thymine dimer lesion can therefore be used to determine theproficiency of nucleotide excision repair capacity in the cell and/orsubject. It should be appreciated that more than one kind of lesion canbe used to determine the strength of a DNA repair pathway in a cell.Thus, for instance, both a thymine dimer and cisplatin adducts can beused to evaluate the proficiency of nucleotide excision repair. Itshould also be appreciated that one lesion can be repaired by more thanone DNA repair pathway (See e.g., Table C).

TABLE B DNA repair pathways, representative lesions, and model celllines useful in establishing repair assays. Example of CorrespondingRepair Representative Deficient Proficient Cell Pathway Lesions CellLine Genotype Line Organism Cell Type Nucleotide cys-syn thymine-GM02344 XPA GM01953 Human Lymphoblastoid Excision Repair thynine mutant(NER) cyclobutane pyrimidine dimer Homologous double strand irs1 XRCC3irs1 + pXRCC3 Hamster Fibroblast Recombination break, when a mutant (HR)separate, intact homologous sequence is available Non double strandXR-C1 DNA PKcs XR-C1 + Human Hamster Epithelial Homologous break inabsence mutant Chromosome End Joining of homologous #8 (NHEJ) sequencesMicrohomology double strand BRCA1−/− BRCA1−/− Wild type MEFs MouseEmbryonic Mediated break between MEFs Fibroblasts End Joiningmicrohomologies (MMEJ) Direct Reversal O6- TK6 MGMT−/− TK6 + MGMT HumanLymphoblastoid (DR) methylguanine Base Excision ethenoadenine Aag−/−Aag−/− Aag−/− + Aag Mouse Embryonic Repair (BER) MEFs (complemented)Fibroblasts Mismatch G:G mismatch HCT116 MLH1 HCT116 + Human ColonRepair mutant Human carcinoma Chromosome #3 lnterstrand Psoralen XP42ROXPF C5RO Human Immortalized Crosslink Crosslink mutant FibroblastsRepair

TABLE C Representative lesions and their associated DNA repair pathwayLesion Main Repair Pathways Cys-Syn Thymine-Thymine cyclobutanepyrimidine dimer NER Double strand break HR, NHEJ, MMEJ Single strandbreak BER Base mismatches MMR, BER Single base insertion loops MMRO⁶-methylguanine DR, NER, MMR 1,N⁶-ethenoadenine BER, DR3,N⁴-ethenocytosine BER, DR cisplatin adducts Cross-link repair, NER, HRabasic sites BER UV irradiation (Cyclobutane Pyrimidine Dimers and (6-4)photoproducts) NER Incubation with MNNG (methylated bases) BER, DRIncubation with Chloroacetaldehyde (etheno bases) BER, DR Incubationwith psoralen + UVA irradiation Cross-link repair, HR, NER Incubationwith BCNU Cross-link repair, HR, NER 7,8-Dihydro-8-oxoguanine (8-oxo-G)BER 7,8-Dihydro-8-oxoguanine (8-oxo-A) BER thymine glycol BER uracil BERhypoxanthine BER xanthine BER N7-methylguanine BER N3-methyladenine BERN1-methyladenine BER, DR N1-methylguanine BER, DR N3-methylcytosine BER,DR N7-methyladenine BER 8-methylguanine BER N3-methylguanine BERO²-methylcytosine BER N3-methylthymine DR O²-methylthymine BERO⁴-methylthymine DR, NER 2,6-Diamino-4-hydroxy-5-formamidopyrimidine(FaPy-G) BER 4,6-Diamino-5-formamidopyrimidine (FaPy-A) BER M₁A(malondialdehyde adduct of adenine) NER M₁C (malondialdehyde adduct ofcytosine) NER M₁G (malondialdehyde adduct of guanine) NER5,6-Dihydrothymine BER 5-Hydroxy-5,6-dihydroxythymine BER5-Hydroxymethyluracil BER 5-Hydroxy-5-methylhydantoin BER5-Hydroxy-5,6-dihydrocytosine BER Cytosine glycol BER5,6-dihydroxycytosine BER 5-Hydroxyhydantoin BER Methyltartonylurea BER8,5′-Cyclodeoxyguanosine NER Urea BER C(5)-C(5) thymidine dihydrodimer ?N-formamidourea ? N-(deoxyguanosin-8-yl)-2-aminofluorene NER3-(deoxyguanosin-N²-yl)-N-acetyl-2-aminofluorene NERN-(Deoxyguanosin-8-yl)-N-acetyl-2-aminofluorene NER(+)-anti-benzo[a]pyrene diol epoxide-N²-dG NER8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B₁ NERN-(deoxyguanosin-8-yl)-2-amino-3-methylimidazo[4,5-f]quinoline NER3-(deoxyguanosin-N²-yl)-4-aminoquinoline-1-oxide NERN-(deoxyguanosin-C8-yl)-4-aminoquinoline-1-oxide NER3-(deoxyadenosin-N⁶-yl)-4-aminoquinoline-1-oxide NERMethylphosphotriester (DNA backbone modification) ? O⁶-ethylguanine DR,NER, MMR ethanoadenine BER 1,2-ethenoguanine ? 2,3-ethenoguanine ? Topposite O⁶-methylguanine BER A opposite 8-oxo-G BER Double strandbreaks with variable overhangs and homologies NHEJ, MMEJ, HR Additionalisomers of cyclobutane pyrimidine dimers and (6-4) photoproducts NERProtein DNA adducts NER, ? All permutations of mismatches MMR DNAlesions in the non-transcribed strand Various (mixed lesions) Normalbases opposite lesions Various (mixed lesions) DNA incubated withtemozolomide Various (mixed lesions) DNA incubated with otherchemotherapeutic drugs Various (mixed lesions) DNA treated with ionizingradiation Various (mixed lesions)Processing

In one aspect, the disclosure provides methods of determining DNA repaircapacity in a cell. In some embodiments, the disclosure provides methodsof determining DNA repair capacity in a cell, the method comprisingintroducing one or more DNA repair vectors into a cell, and determiningthe capacity of the cell to process the one or more DNA repair vectorsthereby determining the DNA repair capacity of the cell.

In some embodiments, processing the one or more DNA repair reportervectors comprises modifying a DNA lesion present in the one or more DNArepair reporter vectors. In some embodiments, processing is detected bya change in a fluorescence signal. In some embodiments, processing isdetected by a change in the transcribed sequence of the one or more DNArepair reporter vectors. In some embodiments, processing is detected bya change in the amount of transcribed sequence of the one or more DNArepair reporter vectors.

In some embodiments, the DNA repair pathway that processes the lesion isnucleotide excision repair, homologous recombination, non-homologous endjoining, microhomology mediated end joining, direct reversal, baseexcision repair, mismatch repair or interstrand crosslink repair.

In some embodiments, the lesions that are processed compriseDNA-crosslinks. In some embodiments, the lesions that are processedcomprise DNA alkylation damage. In some embodiments, the lesions thatare processed block transcription. In some embodiments, the lesions thatare processed induce transcription errors.

DNA repair enzymes process DNA lesions, which results in the removal ormodification of the lesion. However, in some instances the lesions arenot repaired before they are encountered by a DNA or RNA polymerase. Oneof three scenarios occurs when a polymerase runs into damage. In a firstscenario the polymerase is not hindered by the lesions and continuestranscription or replication and inserts the correct ribonucleotide ordeoxyribonucleotide in the growing nucleic acid chain. In a secondscenario, the polymerase is blocked from proceeding further, resultingin the stalling of either transcription (in case of an RNA polymerase)or replication (in case of a DNA polymerase). In a third scenario, thepolymerase is not stalled by the lesion but incorporates an incorrectribonucleotide or deoxyribonucleotide resulting in mutations. Lesionsthat result in DNA mutations and transcriptional mutagenesis aredescribed for instance in Bregeon et al. (Nature Reviews Cancer 11,2011, p 218).

In some embodiments, the disclosure provides methods of determining DNArepair capacity in a cell, the method comprising introducing one or moreDNA repair reporter vectors into a cell, and determining the capacity ofthe cell to process the one or more DNA repair reporter vectors therebydetermining the DNA repair capacity of the cell. In some embodiments,the processing is detected by the change in signal of a reporter gene.Thus, in some embodiments, the DNA repair reporter vectors that areintroduced in the cell comprise a lesion and reporter gene. If thelesion does not block transcription the reporter gene is transcribedresulting in expression of the reporter gene. In some embodiments, thereporter gene encodes a non-fluorescent protein, and the propensity of aDNA lesion to induce transcriptional mutagenesis is detected as anincrease in fluorescent signal. If the cell is able to repair the DNAlesion, a decrease in this fluorescence is observed. If the lesionblocks transcription or slows down transcription the amount of expressedreporter gene is reduced. In some embodiments, the reporter gene is afluorescent protein and the propensity of a lesion to bock transcriptioncan be assessed by a decrease in fluorescence signal. Examples oflesions that the change the signal of fluorescent reporter gene afterprocessing of the DNA repair reporter vectors by the cell are providesin Table D. The lesions that lead to a change is reporter signal arepresumed to partly or completely block transcription

TABLE D Change in fluorescence of selected lesions Changes in LesionMain Repair Pathways Fluorescence Cys-Syn Thymine-Thymine cyclobutanepyrimidine NER Yes dimer Double strand break HR, NHEJ, MMEJ Yes Singlestrand break BER No Base mismatches MMR, BER Yes Single base insertionloops MMR Yes O⁶-methylguanine DR, NER, MMR Yes 1,N⁶-ethenoadenine BER,DR No 3,N⁴-ethenocytosine BER, DR No cisplatin adducts Cross-linkrepair, NER, HR Yes abasic sites BER Yes UV irradiation (CyclobutanePyrimidine Dimers and NER Yes (6-4) photoproducts) Incubation with MNNG(methylated bases) BER, DR Yes Incubation with Chloroacetaldehyde(etheno BER, DR Yes bases) Incubation with psoralen + UVA irradiationCross-link repair, HR, NER Yes Incubation with BCNU Cross-link repair,HR, NER Yes

In some embodiments, the disclosure provides methods of determining DNArepair capacity in a cell, the method comprising introducing one or moreDNA repair reporter vectors into a cell, and determining the capacity ofthe cell to process the one or more DNA repair reporter vectors therebydetermining the DNA repair capacity of the cell. In some embodiments,the processing is detected by sequencing part or all of the transcriptsproduced from the DNA repair reporter vectors. Sequencing thetranscripts allows for the determination if an incorrect nucleotide isinserted when RNA polymerase bypasses the lesion. Ribonucleotidesequences may be translated into deoxynucleotide sequences to facilitatesequencing.

DNA Repair Reporter Vectors

In some embodiments, the disclosure provides methods of determining DNArepair capacity in a cell, the method comprising introducing one or moreDNA repair reporter vectors into a cell, and determining the capacity ofthe cell to process the one or more DNA repair reporter vectors therebydetermining the DNA repair capacity of the cell. DNA repair reportervectors comprise one or more lesions and a means for determining if thelesion has been processed. In some embodiments, the means fordetermining if a lesion has been processed is expression of afluorescent reporter gene. In some embodiments, the fluorescent reportergene is a green fluorescent protein. The placement of the lesion and thegene on the vector should be such that the blocking of transcriptionwould prevent the gene from being expressed. In some embodiments, thelesion is in the reporter gene. In some embodiments, the lesion islocated directly upstream of the reporter gene.

It should be appreciated that a reporter gene can also be used toevaluate the mutational aspect of the repair of the DNA lesion. Forinstance, a mutation can be introduced opposite a lesion in a reportergene (e.g., a fluorescent gene) that inactivates the fluorescent abilityof the protein. If a lesion is acted on, a new mutation may beintroduced into the reporter gene restoring its ability to “report”(e.g., show a fluorescent signal).

The lesion can be introduced into the vector through any means. In someembodiments, the lesion is introduced through site specific engineeringof a vector (See e.g., Shrivastav et al., Carcinogenesis 31, 2010, p59). The site specific engineering allows for the introduction of alesion at any desired position in the DNA repair reporter vector. Insome embodiments, multiple site specific lesions are incorporated in thevector (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 lesions or more). If more thanone lesion is incorporated they can be the same lesion or differentlesions (e.g., both O⁶-methyl guanine or O⁶-methylguanine andN⁷-methylguanine). In some embodiments, multiple lesions that arerepresentative for a particular environmental condition or cancerchemotherapy regimen are introduced into the vector. In someembodiments, multiple lesions that are susceptible to processing by thesame DNA repair pathway are introduced into the same vector. In someembodiments lesions are introduced by treating the vector with a DNAdamaging agent (e.g., UV light, cisplatin, or MNNG), resulting in avector with a number of lesions corresponding to the damaging agent. Ingeneral, treating the vector with an increased dose and/or increasingthe time of exposure will result in a higher number of lesions on thevector. It should be appreciated that some DNA damaging agents mayinduce more than one lesion (e.g., DNA alkylation agent will likelyinduce a number of different alkylated DNA lesions).

In one aspect, DNA repair reporter vectors comprise one or more lesionsand means for determining if the lesion has been processed. In someembodiments, the means for determining if a lesion has been processed isa first nucleic acid sequence that can be sequenced. By sequencing anucleic acid sequence at the site of the lesion or, in some instances,immediately upstream of the lesion, the presence of transcriptionalmutations can be determined. Determining of the sequence allows for thedetermination of the lesion that has been processed by DNA repair. Insome embodiments, the DNA repair reporter vector has both a reportergene (e.g., a fluorescent gene) and a first nucleic acid sequence to besequenced. By assaying both the amount of reporter gene and the sequenceof the first nucleic acid, the DNA repair of the lesion can be evaluatedboth in the context of the blocking of transcription and theincorporation of mutations.

In some embodiments, the DNA repair reporter gene also comprises asecond nucleic acid sequence (or additional nucleic acid sequences). Thesecond (or additional) nucleic acid sequences allow for the coding ofthe DNA reporter repair vector. For instance, the second (or additional)nucleic acid sequences can code for the lesion that was introduced onthe vector and/or the cell-line into which the vector was introduced,and/or the subject from whom the cell originated and/or the particularexperiment or experimental condition.

In some embodiments, the DNA repair reporter vector comprises an originallowing for replication of the vector inside the cell. Having an originon the vector allows for the study of DNA repair in the context ofreplication.

In one aspect, the disclosure provides DNA repair reporter vectors forthe methods, compositions, and kits for determining DNA repair capacityin a cell. In some embodiments, the DNA repair reporter vector comprisesa DNA lesion and a fluorescence reporter gene. In some embodiments, theDNA repair reporter vector comprises a DNA lesion and a first nucleicacid sequence allowing for the identification of the DNA lesion. In someembodiments, the DNA repair reporter vector comprises a DNA lesion and afirst nucleic acid sequence allowing for the identification of the DNAlesion and a fluorescence reporter gene. In some embodiments, the DNArepair reporter vector further comprises a second nucleic acid sequenceallowing for the identification of the DNA repair reporter vector. Insome embodiments, the first nucleic acid sequence allows for thedetermination of the processing of the DNA lesion. In some embodiments,the processing is detected by a change in the transcribed sequence ofthe nucleic acid sequence. In some embodiments, the processing isdetected by a change in the amount of transcribed sequence of thenucleic acid sequence.

In some embodiments, the DNA repair reporter vectors comprise a DNAlesion that is susceptible to processing by nucleotide excision repair,homologous recombination, non-homologous end joining, microhomologymediated end joining, direct reversal, base excision repair, mismatchrepair or interstrand crosslink repair. In some embodiments, the DNArepair reporter vector comprises a DNA lesion that is representative ofa cancer treatment regimen. In some embodiments, the DNA repair reportervector comprises a DNA lesion that is representative of an environmentalcondition.

A DNA repair reporter vector as used herein refers to a nucleic acidvector that can be introduced into a cell to determine the DNA repaircapacity of the cell. As used herein, a “vector” may be any of a numberof nucleic acids into which a desired sequence may be inserted. Vectorsare typically composed of DNA. Vectors include, but are not limited to,plasmids. A desired DNA sequence, such as a reporter gene, may beinserted by restriction and ligation such that it is operably joined toregulatory sequences and may be expressed as an RNA transcript. Vectorsmay further contain one or more marker sequences suitable for use in theidentification of cells which have or have not been transformed ortransfected with the vector. Markers include, for example, genesencoding proteins which increase or decrease either resistance orsensitivity to antibiotics or other compounds, genes which encodeenzymes whose activities are detectable by standard assays known in theart (e.g., β-galactosidase or alkaline phosphatase), and genes whichvisibly affect the phenotype of transformed or transfected cells. TheDNA repair reporter vector may be introduced into an appropriate hostcell by any of a variety of suitable means, e.g., transformation,transfection, conjugation, protoplast fusion, electroporation, calciumphosphate-precipitation, direct microinjection, and the like.

Multiplexing

In one aspect, the disclosure provides methods, compositions and kitsfor determining the activity of multiple DNA repair pathways in multiplecells or subjects in one assay. Prior to the instant disclosure it wasnot possible to adequately determine the activity of multiple DNA repairpathways. Prior to the instant disclosure it was not possible toadequately determine the activity of multiple DNA repair pathways inmultiple cells or subjects. In some embodiments, the multiple DNA repairreporter vectors comprise lesions susceptible to processing bynucleotide excision repair, homologous recombination, non-homologous endjoining, microhomology mediated end joining, direct reversal, baseexcision repair, mismatch repair or interstrand crosslink repair.

In one aspect, the disclosure provides a method of determining multipleDNA repair capacities in a cell, the method comprising introducingmultiple DNA repair reporter vectors into a cell, and determining thecapacity of the cell to process the multiple DNA repair reporter vectorsthereby determining multiple DNA repair capacities in the cell. In someembodiments, the multiple DNA repair reporter vectors comprises at leasttwo, at least three, at least four, at least five, at least six, atleast seven, at least eight, at least nine, at least ten or more DNArepair reporter vectors. In some embodiments, the multiple DNA repairreporter vectors comprise at least two DNA repair reporter vectors. Insome embodiments, the multiple DNA repair reporter vectors comprises atleast four DNA repair reporter vectors.

In some embodiments, if multiple DNA repair reporter vectors areintroduced into a cell, they will each have a unique identifier. Thus,for instance, each DNA repair reporter vector may have a differentfluorescent vector (Such as DsRed, EGFP, EYFP, SCFP, EBDP).Alternatively, or in addition, each DNA repair reporter vector may havea unique nucleic acid sequence to identify the DNA repair reportervector (e.g., by sequencing).

In some embodiments, each DNA repair reporter vector of the multiple DNArepair reporter vectors comprises a unique DNA lesion. In someembodiments, the multiple unique DNA lesions are representative of aspecific cancer treatment regimen. Thus, for instance, multiple uniquelesions caused by an alkylating agent can be introduced is a cell toassess the ability of the cell to repair such lesions. In someembodiments, the multiple unique DNA lesions are representative of amultiple cancer treatment regimen. Thus, for instance, DNA repairreporter vector with lesions representative of alkylating agent therapy,cisplatin therapy and radiation therapy can be introduce in a cell toevaluate how the cell would respond to these different cancer treatmentregimens.

In some embodiments each DNA repair reporter vector of the multiple DNArepair reporter vectors comprises a specific number of DNA lesions. Insome embodiments, it may be desired to know how a cell line wouldresponds with increasing doses of DNA damaging agents, such as in thecase of cancer chemotherapy. Dose responses can be evaluated byassessing multiple DNA repair reporter vectors comprising a specificnumber of DNA lesions. Different number of lesions can be introduced inthe vector, for instance, by exposing the vector to a DNA damaging agentfor different amounts of time. In some embodiments, each DNA repairreporter vector of the multiple DNA repair reporter vectors comprises anumber of DNA lesions corresponding to a specific dose of damagingagent. In some embodiments, each DNA repair reporter vector of themultiple DNA repair reporter vectors comprises a unique identifier.

Kits

In one aspect, the disclosure provides kits for determining the DNArepair capacity in a cell or multiple cells. In some embodiments, thekit comprises one or more DNA repair reporter vectors and instructionsfor use of the one or more DNA repair reporter vectors. Instructions foruse in the kits include instructions for any aspect of the methodsdescribed herein, such as transfection of the plasmids into the cells,reporter assays and sequencing of the DNA repair reporter transcripts.In some embodiments, the kit comprises at least two, at least three, atleast four, at least five, at least six, at least seven, at least eight,at least nine, at least ten or more DNA repair reporter vectors. In someembodiments, the kit comprises at least two DNA repair reporter vectors.In some embodiments, the kit comprises at least four DNA repair reportervectors. In some embodiments, each DNA repair reporter vector of the kitcomprises a unique DNA lesion. In some embodiments, each DNA repairreporter vector of the kit comprises a unique number of DNA lesions. Insome embodiments, each DNA repair reporter vector of the kit compriseslesions susceptible to processing by nucleotide excision repair,homologous recombination, non-homologous end joining, microhomologymediated end joining, direct reversal, base excision repair, mismatchrepair or interstrand crosslink repair.

In one aspect, the disclosure provides kits for determining the DNArepair capacity of a particular DNA repair pathway in a cell or multiplecells. In some embodiments, the DNA repair pathway is nucleotideexcision repair, homologous recombination, non-homologous end joining,microhomology mediated end joining, direct reversal, base excisionrepair, mismatch repair or interstrand crosslink repair.

In one aspect, the disclosure provides kits for determining thepropensity of a subject to respond to a cancer treatment regimencomprising one or more DNA repair reporter vectors, wherein the one ormore DNA repair reporter vectors comprise lesions representative of acancer treatment regimen, and instructions for use of the one or moreDNA repair reporter vectors.

In one aspect, the disclosure provides kits for determining thesusceptibility of a subject to an environmental condition comprising oneor more DNA repair reporter vectors, wherein the one or more DNA repairreporter vectors comprise lesions representative of an environmentalcondition, and instructions for use of the one or more DNA repairreporter vectors.

In one aspect, the disclosure provides kits for determining the repaircapacity of a cell line comprising one or more DNA repair reportervectors, wherein the one or more DNA repair reporter vectors allow forthe determination of the repair capacity of the cell line, andinstructions for use of the one or more DNA repair reporter vectors.

In some embodiments, the kit further comprises a cell line with a knownDNA repair capacity. The kit may also include additional components,such as vials, solutions, buffers, plates, and reagents to perform themethods disclosed herein.

In one aspect, the kits allow for a high throughput, comprehensive, andquantitative assessment of the capacity of cells to repair DNA damagevia a large number pathways in a single assay. DNA repair capacityvaries among individuals, and deficiencies are associated with a largenumber of diseases. The kits serve as a diagnostic for suchdeficiencies. Sensitivity to DNA damaging agents (includingchemotherapeutic and other drugs, sunlight, ionizing radiation,cigarette smoke, as well as endogenous sources of DNA damage) variessignificantly among healthy individuals. These differences are, at leastin part, a consequence of inter-individual differences in DNA repaircapacity. The kit also provides a means of offering personalized diseaseprevention to individual patients with particular DNA repairdeficiencies or abnormalities. In addition, the kit can be used tooptimize treatment of known diseases. For example, an ideal chemotherapycould be tailored to an individual patient based on differences in theDNA repair capacity of the cancer patient's healthy normal tissues, ascompared with that of the tumor.

In some embodiments, measurements of DNA repair capacity are based onthe transient transfection of cells with a library of plasmid DNAreporters containing different types or doses of DNA damage. Anembodiment of a DNA repair reporter vector is provided in FIG. 15.

Detection of reporters is achieved by a number of methods. In a firstmethod of detection, repair capacity is measured in intact cells viafluorescence-based host cell reactivation technology, wherein repair oftranscription blocking DNA damage results in measurable reactivation ofotherwise inhibited fluorescent protein expression. Fluorescence in twoor more channels is measured, for instance, by flow cytometry, or laserscanning cytometry. Each fluorescent reporter color corresponds torepair of a different DNA lesion, or a different dose of DNA damage. Onefluorescent reporter is used as a transfection control. A fluorescentviability stain can be used to exclude dead cells, or a fluorescentnuclear stain can be used to analyze repair in a cell cycle dependentmanner. An example of how the “Lumens” technology can be applied tohuman cells is illustrated in FIGS. 16-19).

The given method of measuring DNA repair capacity combines theefficiency of high throughput transfection with the speed, sensitivity,and multiplexing capability of flow cytometric analysis to yield arapid, high throughput, multiplexed host cell reactivation assay.

In a second method of detection instead of measuring fluorescenttranslation products of transcription reporters, mRNA transcriptsthemselves are measured and analyzed. (A kit according this technologyis called “Sequens” in FIG. 14. The two methods are complimentary, witheach having unique advantages (Table E).

TABLE E comparison of fluorescent and sequencing methods FlourescenceSequencing Advantages Straight-forward procedure (electroporation, Can‘count’ relative numbers of flourescent plasmid preparation) andanalysis protein transcripts Single-cell resolution Data ontranscription errors/deletions/ Binary Results frameshift mutationsInexpensive Applicable to most DNA lesions Can compare results to FACSdata Disadvantages Not all damage inhibits RNA polymerase No single-cellresolution Lose information about transcription errors Longer samplepreparation without immediate (deletions, RNA base misincorporation,results frameshifts) and aborted or improperly-folded More expensivetranscripts Works better with site-specific lesions Potentially moredifficult data analysis

An experiment analogous to that described for fluorescent reportersabove includes reporter transcription measured directly by countingtranscripts produced when cells are transfected with either damaged orundamaged plasmids. Direct detection of transcripts provides additionalinformation on the DNA repair capacity of the cell or subject. Whereasthe optical properties of fluorescent reporters currently limit thenumber that can be simultaneously and independently detected to 5 or 6,thousands of unique reporter transcripts (or more) can be measuredsimultaneously using high throughput DNA sequencing. A sequencing basedassay for DNA repair capacity is provided in FIGS. 20 and 21 (See alsoTable F).

TABLE F Sequencing HCR. Number of Number Colonies of EGFP Number ofSample cDNA Sequenced Colonies RFP Colonies RFP Undamaged 20 14 6 RFP at800 J/m2 12 11 1

In a low-throughput sequencing HCR, sequences from reporters damagedwith UV radiation were represented less often among cDNA clones,consistent with reduced transcription relative to the undamagedcontrols. Next generation sequencing of the same materials provides thestatistical power needed to resolve differences in repair capacity.

Although sequencing based repair assays can in principle be approachedusing randomly damaged plasmids, knowing the exact location of thelesions in advance greatly increases signal to noise and focuses theanalysis to a short region of the reporter gene. An example of how sitespecifically introduced lesions are applied in a DNA repair assay isgiven in FIG. 23, and the methodology developing for the production ofthe necessary reporters is presented in FIG. 24.

In one aspect, the sequencing based DNA repair capacity kit provides anew technology (FIGS. 20-26) for a new method for measuring DNA repaircapacity. In some embodiments, a library of site-specific reporters inclose proximity to the identifying bar code that corresponds to eachrespective DNA lesion is provided. This proximity is constrained by themaximum read length of the high throughput sequencing equipment,currently about 150 nucleotides on most instruments. The scope of thesubstrates in the Sequens kit is described in FIG. 27.

Existing technology restricts measurements of DNA repair capacity to asingle type of DNA damage in each assay. The most direct comparisons canbe made to HCR assays that are done with chloramphenicolacetyltransferase reporters. However CAT based assays require celllysis, several hours of manual sample processing, one-at-a-time, involveradioactive tracers, and there is no way to control for transfectionefficiency. Luciferase has also been used as a reporter in HCR assays,however this method is limited to only two colors, and also requirescell lysis. Fluorescent reporters have been described in a few papers inthe literature, however only two colors at a time have been used, and norigorous method of detection with appropriate controls and carefulcalibration against previously characterized methods has been reported.The examples in the literature also do not describe methods of scalingup the procedure for high throughput 96-well formats. The kits providedherein enable repair of at least 4 (Lumens) and up to 100s or even 1000sor more (Sequens) types of damage or doses of damage to be assayedsimultaneously. The use of next generation sequencing to detect reportertranscripts as reporters for DNA repair capacity has not been previouslyreported. Site specific DNA damage has not been used in a massivelyparallel analysis as provided herein. Previous methods generally requiresamples to be handled and analyzed one at a time, whereas the methodsprovided herein enable one to assay multiple samples simultaneously andautomatically using robotics (in the case of flow cytometry) or barcodes and software (for sequencing based detection). Previous methodsmake use of variable reporter constructs, readouts and methods oftransfection, potentially confounding comparisons among experimentsconducted in different laboratories. Rigorous controls for theseconfounding variables are generally not carried out. Inter-laboratoryvariability, together with the cumbersome standard approaches tomeasuring DNA repair capacity make them low throughput, and thusrefractory to large scale epidemiological studies and clinicalapplications. The high-throughput kits provided herein establishrigorous controls for transfection efficiency, variability in reporterexpression, and possible inter-laboratory variability (using a controlcell line against which samples can be compared). A particular advantageof the sequencing-based detection system is the possibility of shippingnucleic acid samples to an off-site location for analysis by a thirdparty; existing methods require the analysis of intact cells or freshcell lysates. Previous methods also require significant background onthe part of the user. The methods provided herein make DNA repaircapacity measurements accessible to scientists with basic laboratorytraining. The Lumens kit provides a comprehensive spectrum of DNA repaircapacity for any cell culture amenable to transient transfection inabout 24 hours. Embodiments of kits of the invention are provided below

QUANTREPAIR® “Lumens” (Cytometry Based Approach)

-   -   A library of 5 or more undamaged fluorescent reporter encoding        plasmids    -   A library of 4 or more fluorescent reporter encoding plasmids        that contain DNA lesions    -   Buffers    -   8 peak fluorescent beads to calibrate flow cytometers    -   A control cell line against which to validate and compare        results    -   Data analysis templates    -   A manual describing how to use the kit and analyze data        QUANTREPAIR® “Sequens” (Sequencing Based Approach)    -   A library of 10 or more undamaged reporter plasmids    -   A library of 10 or more bar-coded reporter plasmids bearing        site-specifically incorporated lesions    -   Buffers    -   Primers for cDNA synthesis and amplification    -   A control cell line against which to validate and compare        results    -   Data analysis templates    -   A manual describing how to use the kit and analyze data

The kit may be provided either as a research tool or as a clinical test.In the research laboratory, the kit may provide a rapid and reliable wayto measure DNA repair capacity/deficiency in previously uncharacterizedcell lines. It may also be used in conjunction with previouslyuncharacterized types of DNA damage to determine whether they arerepaired in cells. In the clinical setting, the assay may be used as aroutine blood test for an individual's repair capacity spectrum, and thedata used to advance personalized prevention and treatment of disease.

The present invention is further illustrated by the following Examples,which in no way should be construed as further limiting. The entirecontents of all of the references (including literature references,issued patents, published patent applications, and co-pending patentapplications) cited throughout this application are hereby expresslyincorporated by reference, in particular for the teaching that isreferenced hereinabove.

EXAMPLES Example 1: HT-HCR and HCR-Seq: High Throughput Tools for DNARepair Capacity Measurements

Materials and Methods

Human Cells

A total of 19 human cell cultures representative of 12 individuals wereobtained from the Coriell Institute (Table 1). Epstein Barr virustransformed lymphoblastoid cell lines were maintained in log phase inGIBCO RPMI 1640 supplemented with 15% fetal bovine serum (FBS), in thepresence of 2 mM L-glutamine, penicillin and streptomycin. Primary skinfibroblasts were cultured in GIBCO DMEM/F12 media supplemented with 15%FBS and, in the presence of 2 mM L-glutamine and antibiotics.Fibroblasts were subcultured by trypsinization.

Plasmids

Plasmids containing genes encoding the fluorescent proteins AmCyan,EGFP, mOrange, and mPlum were purchased from Clontech, and a plasmidencoding tagBFP was purchased from Axxora. Reporter genes were subclonedinto the pmax cloning vector (Lonza) between the KpnI and SacIrestriction sites in the multiple cloning site. The Kozak translationinitiation consensus sequence and an additional NheI restriction sitewere introduced at the 5′ end of each reporter, and a HindIIIrestriction site was added to the 3′ end. The pmax cloning vector placesreporter genes under a CMV Intermediate-Early promoter. Plasmids wereamplified using E. coli DH5-alpha, and purified using Qiagenendotoxin-free giga kits. Constructs were confirmed by DNA sequencingand restriction digests.

UV-Irradiation

Plasmids were irradiated at a concentration of 50 ng/uL in a volume of1.5 mL in 10 cm polystyrene petri dishes (without lids) with UVC lightgenerated by a STRATALINKER® 2000 box. Following treatment, reporterplasmids were combined in the following ratio: 1 part tagBFP, 10 partsAmCyan, 1 part GFP, 2 parts mOrange, and 4 parts mPlum. Increasedamounts were used to compensate for the weaker fluorescence intensitiesobserved for some of the reporters. Corresponding mixtures of plasmidswithout UV irradiation were prepared from the same solutions, exceptwithout treatment. Although experiments performed with the mixturecontaining irradiated plasmids have been labeled “damaged”, and thosecontaining untreated plasmids have been labeled “undamaged”, everytransfection included a transfection reporter, which always remainedundamaged. The transfection reporter was used to normalize for potentialvariation in transfection efficiency. Further details regarding the dosedelivered to each plasmid are available in Table 2. Plasmid mixtureswere ethanol precipitated, and washed with 70% ethanol. Pellets weredissolved in TE buffer for 30 minutes at room temperature, to a finalconcentration of approximately 1.5 micrograms per microliter. Thedamaged and undamaged solutions were adjusted to the same finalconcentration. DNA concentrations were verified using a Nanodropspectrophotometer.

Transfection

Lymphoblastoid cells at a concentration of 2×10⁷/mL (total volume, 100uL) in complete media were combined with 9 micrograms of reporterplasmid mixtures using the cocktails described above. The mixtures wereelectroporated using a Bio-rad MXCELL™ gene pulser, with an exponentialwaveform at 260V and 950 uF. Following electroporation, cells werediluted 5-fold in fresh culture media, and divided into three 96-wellplates. Cells were incubated at 37° C., 5% CO₂, and one plate wasremoved for analysis at each time point (18 and 40 hours). The thirdplate was reserved as a backup. Each transfection was performed induplicate, on three separate days. The same procedures were used for thesamples that were analyzed by sequencing, however each transfection wasperformed in quadruplicate, the quadruplicate transfections were dilutedinto a single 2 mL culture.

Flow Cytometry

Live cells suspended in culture media were analyzed for fluorescence ona BD LSRII cytometer, running FACSDIVA™ software. Cell debris, doubletsand aggregates were excluded based on their side scatter and forwardscatter properties. TOPRO®-3 was added to cells 5-10 minutes prior toanalysis, and used to exclude dead cells from the analysis. Thefollowing fluorophores and their corresponding detectors (parentheses)were used: tagBFP (Pacific Blue), AmCyan (AmCyan), EGFP (FITC), mOrange(PE), mPlum (PE-Cy5-5), and TOPRO-3 (APC). The linear range for thecorresponding photomultiplier tubes was determined using BD Rainbowfluorescent beads and unlabeled polystyrene beads based on the signal tonoise ratio, % CV, and M1/M2 parameters as previously described (REF).Compensation was set using single color controls. Regions correspondingto cells positive for each of the 5 fluorescent proteins wereestablished using single color dropout controls.

Fluorescence signal (F) was computed using equation 1:

$\begin{matrix}{F = \frac{N \times {MFI}}{S}} & (1)\end{matrix}$

Where N is the total number of cells appearing in the positive regionfor that fluorophore, MFI is the mean fluorescence intensity of the Ncells, and S is the total number of live cells measured in theexperiment. The fluorescence signal of an undamaged plasmid included inall transfections to control for transfection efficiency was designatedF^(E). The normalized fluorescence signal for a given reporter F^(O) wascalculated using equation 2:

$\begin{matrix}{F^{O} = \frac{F}{F^{E}}} & (2)\end{matrix}$

Normalized reporter expression from a damaged reporter plasmid, F^(O)_(dam), and that from the same reporter plasmid in the absence ofdamage, F^(O) _(un), were used to compute the percent reporterexpression (% R.E.) using equation 3:

$\begin{matrix}{{\%\mspace{14mu}{R.E.}} = \frac{F_{{da}\; m}^{O}}{F_{un}^{O}}} & (3)\end{matrix}$Preparation of Plasmids Containing a Site-Specific Thymine Dimer

A site-specific thymine dimer was introduced into the GFP reporterplasmid using methods described previously [21]. Briefly, two nickingsites for the enzyme Nb.Bpu10I in the GFP reporter gene near the 3′ endwere used to excise a single stranded oligonucleotide of 18 bp inlength: 5′-TCAGGGCGGATTGGGTGC-3′ (SEQ ID NO: 6). The nicking sites flanka silent mutation that has been introduced to generate a TpT sequence inthe transcribed strand of the plasmid. A synthetic oligonucleotide5′-TCAGGGCGGAT< >TGGGTGC-3′ (SEQ ID NO: 7), containing a thymine-thyminecis-syn cyclobutane dimer (indicated by T< >T) and synthesized byTriLink BioTechnologies using a cis-syn thymine dimer phosphoramidite(Glenn Research) was ligated into the gapped plasmid. The incorporationof the site-specific thymine dimer in the plasmid was confirmed by anendonucleolytic digest with the bifunctional enzyme thymine dimerspecific glycosylase/AP lyase (T4 PDG).

Isolation of Total RNA for RNAseq

At 18 hours, transfected cells were harvested by centrifugation, washedthree times with fresh media, and resuspended in 1 mL TRIZOL® reagent.The suspension was extracted with 200 μL chloroform. The aqueous phasewas removed, combined with one volume of absolute ethanol, and appliedto a Qiagen RNEASY® mini-prep spin column. The column was then washedtwo times with 500 μL buffer RPE (Qiagen), and finally eluted in 40 μLdiethylpyrocarbonate (DEPC) treated water. From this point forward, RNAwas handled in Eppendorf DNA LoBind tubes to minimize loss of material.The quality of the RNA preparation was determined using a bioanalyzer toconfirm a RIN of at least 9.0. 1 μg of total RNA was stored in TE Bufferat −80° C. until submission for RNAseq.

Isolation of mRNA and Synthesis of cDNA

From the remaining total RNA, mRNA was isolated using a Qiagen Oligotexkit, using the manufacturer's protocol, but substituting Eppendorf DNALoBind tubes for those provided with the kit. In the final step, mRNAwas eluted in 20 uL buffer OEB preheated to 70° C. 5 μL of the eluatewas transferred to a LoBind tube, combined with 1 μL of DNAse buffer and1 unit of DNAseI (Invitrogen). The mixture was brought up to a 10 μLvolume with DEPC treated water, and incubated for 15 minutes at roomtemperature. DNAse was inactivated by addition of 1 μL of 25 mM EDTA,followed by incubation at 65° C. for 10 minutes. A cocktail comprised of2×RT buffer (Qiagen), oligo-dT(12-18) (125 ng/uL; invitrogen), 4 unitsof RNAse inhibitor (Qiagen), 5 mM dNTPs, and 4 units of reversetranscriptase (Omniscript; Qiagen) was prepared, and 8 μL added to theDNAse digest. The reaction was incubated for 1 hour at 37° C. No-RTcontrols were performed identically, except for the exclusion of thereverse transcriptase.

Specific Amplification of Reporter cDNA by PCR

cDNA samples were amplified with primers specific to the 3′ and 5′ UTRregions of the pMax vector. The following primers were synthesized forspecific amplification of reporter cDNA:

(SEQ ID NO: 1) 5UTR: 5′-TTG CTA ACG CAG TCA GTG CT-3′ (SEQ ID NO: 2)3UTR: 5′-GCA TTC TAG TTG TGG TTT GTC C-3′

1.5 μL of cDNA was PCR amplified in a 25 μL reaction volume with 1×PCRbuffer (Denville), 0.5 μM primers, 0.2 mM dNTPs, and 1 unit Taqpolymerase (Denville). Specific amplification was confirmed by gelelectrophoresis and analysis on a bioanalyzer chip. Water and EGFPencoded plasmids were used as negative and positive controls,respectively. Finally, reactions were cleaned up using a Qiagen PCRcleanup kit according to the manufacturer's protocol, and eluted in 50uL of TE.

Fragmentation of DNA

250 ng of PCR product was diluted to a total volume of 130 uL in TEbuffer. The DNA was fragmented in a Covaris microTUBE using a Covaris S2sonicator (Duty Cycle 10%, Intensity 5, 200 cycles per burst, 180seconds. Fragmentation to a target base pair peak of 150 bp was checkedusing a Agilent BioAnalyzer.

Next Generation Sequencing

Total RNA and fragmented DNA samples were submitted to the MITbiomicrocenter core facility for preparation and sequencing. Briefly,total RNA was poly-A purified and converted to cDNA DNA using theIllumina Tru-Seq protocol. Library construction from cDNA and fragmentedDNA was performed using the Beckman Coulter SPRI-works system (REF).During library amplification, a unique bar-code was introduced for eachof 8 samples corresponding to the four transfections (#1953 undam, #1953dam, #2344 undam, #2344 dam), from which both total RNA and PCRamplified reporter cDNA were generated. Samples were clustered on asequencing lane and run on an Illumina HISEQ™ 2000 instrument. Imageanalysis, base calling and sequence alignment to a synthetic genomeconsisting of the human genome and the five fluorescent reporter geneswere performed using the Illumina Pipeline.

Next Generation Sequencing Data Analysis

Both RNAseq and DNAseq data were analyzed using the Tuxedo softwaresuite. Reads were aligned to the human genome and the five reporter genesequences, and junction reads determined. Additional details of allanalyses including input parameters are available in supplemental TableS1. Cufflinks was run to quantify reads in terms reads per kilobase ofexon model per million mapped reads (RPKM) [18]. Single nucleotidemutations, as well as insertions and deletions (indels) present in theRNAseq and DNAseq data were identified using the software packageVarScan [19]. The software required a minimum read depth of 8, and atleast 2 reads supporting a mutation at a given position. Variants werereported if they were detected in at least 1% of all reads covering agiven position, or at least 2 unique reads after removal of duplicates.Variants appearing in the first 168 nucleotides were excluded fromfurther analysis because this region includes the chimeric intron andthe binding site of the 5′ UTR primer. Likewise, the variants appearingin the terminal 22 nucleotides were excluded because this sequencederives from the 3′ UTR primer. Variants observed at the positioncorresponding to the site-specific thymine dimer prompted a targetedsearch for similar variants in transcripts expressed from randomlydamaged reporters. Scripts were used to generate a list of all deletions6 bases or longer and spanning an ApA sequence, and appearing at afrequency of at least 0.01% of all reads. The global frequency of themost commonly observed base substitutions opposite the thymine dimer(5′A→G) and (3′A→T), as well as the dinucleotide mutation (AA→GT), wasdetermined for all ApA sequences for each sample.

Results

A high throughput multicolor host cell reactivation assay (HT-HCR) wassuccessfully deployed in 19 cell lines from 12 individuals (Table 1).Because multiple cell types were studied from some individuals, Greekletters (α-η) have been assigned to refer to the individuals from whomthe cells were derived. Electroporation yielded consistently hightransfection efficiency ranging between 10 and 50% in all cells studied.Chromatin immunoprecipitation of DNA isolated from transfected cellsusing antibodies to histone H3 or H4 confirmed the chromatinization ofreporter plasmids (FIG. 8). Expression levels of five fluorescentreporter proteins were quantitated simultaneously and independentlyusing flow cytometry. In addition, a dead cell stain (TOPRO®-3) wassuccessfully used to exclude dead cells from flow cytometric analysis.Use of 96-well electroporation plates reduced the time required fortransfection to less than one minute per sample, and use of a BD HighThroughput Sampler permitted data acquisition in less than 10 minutesactive time.

In vitro treatment of plasmids with UVC light resulted in adose-dependent reduction in reporter expression. When each of the fivefluorescent reporter plasmids was treated with a unique dose of UVC(Plasmid combination #1 in Table 2), and subsequently co-transfectedinto cells, a dose-response curve was generated from a single experimentthat requires only two transfections (FIG. 1). Dose-response curvesspanning up to 3 decades of percent reporter expression (% R.E.) wereobtained for 7 lymphoblastoid cell lines (FIG. 2), chosen because theyhave been characterized for their capacity to repair UV-irradiated DNApreviously by another method [8]. Two cell lines were derived fromhealthy individuals, and five from xeroderma pigmentosum patients withknown genetic defects in the NER pathway (Table 1). Differences inrepair capacity were most pronounced at the highest dose to plasmid (800J/m²), with % R.E. values varying over a range of about 100-fold amongthe cell lines. As expected, the highest repair capacity was observedfor lymphoblastoid cell lines derived from apparently healthyindividuals. Moderately reduced repair was observed for two XPC celllines, and a severe defect was evident for XPA and XPD cell lines.Between 18 and 40 hours, % R.E. increased for most cell lines (FIG. 2),consistent with time-dependent repair of transcription blocking lesions.

The HT-HCR data presented in FIG. 2 reproduce those from literature [8].In that study, chloramphenicol acetyl transferase (CAT) expression wasused as the reporter. Two complementary methods were used to compare ourdata to those in the literature. First, the percent CAT expression (%CAT) reported at a single dose of UV irradiation (300 J/m² in the Athaset al. study) was found to be highly correlated with % R.E. at a singledose (400 J/m²) in the present study (R²=0.92, p=0.0006). The relativerepair capacity of multiple cell lines has also been compared bycalculating the parameter D_(o), which corresponds to the dose at whichthe HCR dose response curve falls below 37% reporter expression [20].D_(o) was calculated from our experimental data and was also found to behighly correlated with the literature values (R²=0.92, p<0.0001).

To confirm that the dose response curves in FIG. 2 could be obtainedindependent of the choice of fluorescent reporters, the experiment wasrepeated with the plasmids shuffled so that each received a differentdose (Plasmid combination #2 in Table 2). The pattern of dose responsecurves and the relationship to the data in FIG. 2 are presented FIG. 3.Once again, repair capacity measured in % R.E. at the highest dose toplasmid (800 J/m²) varied over a range of about 100-fold among the celllines, and % R.E. increased between 18 and 40 hours (FIGS. 3a and 3b ).Our assay again reproduces the literature data (FIG. 3c ). Repaircapacity measurements from the two combinations of reporter plasmidswere highly correlated (FIG. 3d ).

HT-HCR assays were also carried out on 7 primary untransformed skinfibroblasts and Eppstein-Barr virus transformed lymphoblastoid celllines derived from the same individuals (represented as α-η in Table 1).These experiments included cells from 4 apparently healthy individuals,and 3 XP patients. A similar pattern of dose response curves wasobtained for both fibroblasts and lymphoblastoid cells (FIG. 4).Overall, absolute NER capacity measured in fibroblasts appeared to besomewhat higher than that in the lymphoblastoid cell lines, however therelative differences in repair capacity among individuals were largelypreserved in the two cell types. Comparison of repair capacity measuredat 800 J/m² indicated that NER phenotype is strongly correlated betweenthe two cell types (R²=0.94, p=0.0003).

A site-specific thymine dimer spanning positions 614-615 of the GFPsequence was successfully introduced into transcribed strand of the pmaxGFP reporter plasmid using previously described methods [21]. Most ofthe plasmids were nicked upon incubation with the bifunctional thyminedimer specific DNA glycosylase/AP lyase, T4 PDG (FIG. 5), indicatingthat nearly all of the material contained the lesion. Followingtransfection into cells, GFP expression from the lesion-containingplasmid was reduced relative to that from an undamaged reporter. Asexpected, the largest inhibition of reporter expression was observed inNER deficient cells.

Two cell lines exhibiting a large difference in their NER capacity(GM02344 and GM01953) were selected for comparative repair capacitymeasurements by HT-HCR and HCRseq. The two cell lines were transfectedwith plasmid combination #3 in Table 2. An aliquot of cells was removedfrom the transfection at 18 hours for flow cytometric analysis, and theremaining cells were solubilized in Trizol reagent for subsequentanalysis by HCRseq (see below). GFP expression from the thymine dimercontaining reporter plasmid was successfully measured followingco-transfection with additional reporter plasmids that had been randomlydamaged with several doses of UV radiation (FIG. 11). The dose responsecurves generated from the randomly damaged plasmids are presented inFIG. 6 b.

For HCRseq analysis, total RNA was isolated from the aliquot of cellsthat was reserved in TRIZOL®. The quality of the RNA was checked using abioanalyzer, and the RNA Integrity Number was found to be at least 9.0for all samples. Two separate experiments were then performed. First, analiquot of total RNA was submitted directly for RNAseq analysis. In thesecond experiment, reporter transcripts were enriched by selective PCRamplification. Because these samples were submitted as DNA, thisexperiment and the resulting data are referred to as “DNAseq”. For theDNAseq experiment, total RNA was purified to mRNA and reversetranscribed. PCR amplification of cDNA using primers specific for the 5′and 3′ UTR of the reporter genes generated the expected single ˜800 bpamplicon (FIG. 9). PCR amplification was dependent upon reversetranscription, confirming that the mRNA was free from plasmidcontamination. In both agarose gels and bioanalyzer traces, ampliconsgenerated from cDNA templates were found to migrate slightly ahead ofthose generated from plasmid templates. This was expected based on thepresence of a 136 bp chimeric intron present in the reporter plasmid,and provides further confirmation that mRNA was isolated without plasmidcontamination. Sonication successfully fragmented PCR amplicons to apeak size of approximately 150 bp, as recommended for Illumina TruSeqsample processing.

A total of 180,216,333 reads were generated for 8 multiplexed samples ina single HISEQ™ lane (Table 3). Using the barcode sequences that wereintroduced during sample prep, 92,111,949 (51.1%) of the reads wereassigned to the four samples (A, B, C and D) from the RNAseq experiment,and 88,104,384 (48.9%) were assigned to the DNAseq experiment (E, F, G,and H). Between 15 and 25 million reads were assigned to each of the 8samples. Additional alignment statistics are available in supplementalTables S2-S5.

Relative expression levels were determined for both endogenous andreporter transcripts using the RNAseq data. (Results of the DNAseqexperiment are discussed below). Reporter transcripts were found to beamong the most highly expressed genes (FIG. 10), representingapproximately 1.7% of the total reads. As expected, reporter expressionfrom UV-treated plasmids was reduced in a dose-dependent manner (FIG. 6c). The dose response for the XPA cell line (GM02344) was more pronouncedthan that for the cell line derived from an apparently healthyindividual (GM01953), mirroring the pattern of dose response curvesobtained when reporter expression for the same samples was estimatedusing flow cytometry (FIG. 6b ). Reporter expression from plasmidscontaining a site-specific CPD in the transcribed strand was likewisereduced in an NER-dependent manner (FIG. 11). Very few endogenoustranscripts showed a significant (greater than 2-fold) change inexpression in the presence of UV treated plasmids, and those that weredetected are not known to play an important role in DNA repair(Supplemental Table S6).

Sequence-level analysis of transcripts confirmed a previously reportedmiss-splice in intron 4 of the XPA transcript for cell line GM02344, andrevealed changes in reporter transcripts at the position correspondingto the site-specific CPD in the transcribed strand for both cell lines.Base substitutions and rare deletions were detected in reportertranscripts at the position corresponding to the site-specific CPD (FIG.7). The most frequently observed base change, an A→G mutation at the 3′Adenine in the ApA sequence opposite the CPD, was detected at afrequency of about 1.5% of reads in cells with no known repair defect(GM01953), and at an elevated rate of about 10% of reads in the repairdeficient cells from GM02344 (FIG. 7a ). In transcripts expressed fromthe undamaged plasmid, the frequency of the A→G mutation was less than0.1%. Rare deletions spanning the ApA sequence were detected at afrequency of 0.37% for GM01953, and 1% for GM02344 (FIG. 7b ). Deletionswere not observed at this position in transcripts expressed from theundamaged reporter plasmid. Frequencies for additional sequence changesobserved opposite the site-specific CPD are provided in SupplementalTable S7. Turning to endogenous transcripts, reduced expression and anexpected lack of regular junction reads spanning intron 4 of the XPAgene from GM02344 was observed (FIG. 12), confirming a previouslyreported missplice in XPA transcripts due to the homozygous 555G>Cmutation [22].

Selective amplification of cDNA derived from reporter transcriptsyielded a 50-fold enrichment of reads aligning to reporter sequences,and corroborated results obtained from the RNAseq analysis. Among readsassociated with the DNAseq samples, 74,621,260 (84.7%) aligned to atleast one of the five reporter genes. In order to compare data from theDNAseq experiment directly with those from the RNAseq experiment, RPKMvalues were calculated for both data sets. Dose response curvesgenerated from the DNAseq data recapitulated the trends observed inthose generated from both RNAseq and flow cytometry data (FIG. 6d ).Unexpectedly, expression from the reporter containing a site-specificCPD appeared to be higher in the XPA deficient cell line (GM02344) thanin the one derived from an apparently healthy individual (GM01953). Thepattern of sequence level changes in transcripts at the positioncorresponding to the site-specific CPD was similar to that detected inthe RNAseq data (FIG. 7), however the frequency of deletions was reducedslightly relative to that estimated from the RNAseq data (FIG. 7d ).Rare deletions spanning ApA sequences in reporters from the randomlyUV-damaged reporters were also detected (FIG. 13), with the highestfrequency again observed in reporter isolated from Phoebe. Frequenciesfor additional sequence changes observed opposite the site-specific CPDare provided in Supplemental Table S8.

Discussion

Despite the critical role of DNA repair in preventing disease, methodsof measuring DNA repair capacity have so far lagged behind the demandsthat must be met if such a metric is to be used to personalize theprevention and treatment of cancer and other diseases caused byinefficient repair of DNA damage. We present several new tools thatenable rapid, high throughput measurements of DNA repair capacity forany lesion that affects either the level or the sequence of reportertranscripts.

The flow cytometric HT-HCR method reproduced data collected previouslyfor the same cell lines [8]. By using multiple fluorescent reporters, a96-well format, and automated sample processing, the method is muchfaster and less labor intensive than the standard CAT-based HCR assay.Flow cytometers equipped with a high throughput sampler enablecollection of multiple time points from a single transfection withoutthe requirement for significant additional labor. Furthermore,experimental errors are reduced by co-transfection of reporters, withnormalization to an undamaged control plasmid that is included in everytransfection. Because standard oncology labs are equipped with flowcytometers, the assay can readily be used in a clinical setting. Thus,the HT-HCR removes a major barrier to epidemiological studies of DNArepair capacity that include large populations, and potentially morethan one repair pathway.

We demonstrated an application of the HT-HCR to the question of whetherNER capacity in human lymphoblastoid cells is representative of repaircapacity in other tissues. Lymphoblastoid cells provide a convenientsource of large numbers of cells for use in human variability studies,however the extent to which they represent a faithful surrogate forother cells in primary tissues has been called into question [23-25].The present data indicate a strong correlation between NER capacity inprimary human fibroblasts and the transformed B-lymphoblastoid cells(FIG. 4). The strong correlation further illustrates that the assay canbe carried out reproducibly in cells with disparate morphology.

The use of next generation sequencing to detect reporter transcriptsthemselves (HCR-seq), in place of their fluorescent translationproducts, allows for an increase in throughput, and to measure repairevents that are not readily detected using flow cytometric approaches.We have validated the HCR-seq approach by showing that a similar patternof dose-response curves is obtained for HCR of UV-irradiated plasmids asdetected by three methods (FIG. 6). Whereas HT-HCR allowed for thesimultaneous detection of 5 independent repair reporters, the HCR-seqpermitted the measurement of 40 reporters (5 reporter genes×8bar-codes). In the context of the RNAseq data, 20 of these reporterswere detected at a sufficient coverage to obtain dose response curves(FIG. 6). As these represented less than 1% of the total mapped reads inthe experiment, it can be estimated that at least 2000 reporters (or 200dose-response curves) could be independently assayed on a single lane ifendogenous transcripts were excluded from the assay. The robustness ofthe data after selective PCR amplification demonstrates the feasibilityof such experiments.

The four dose response curves derived from sequencing data and presentedin FIGS. 6c and 6d were acquired at a cost of approximately $750/curve.However, several considerations would reduce the cost ofsequencing-based assays if deployed in large scale population studies.As cost of sequencing continues to fall, and particularly if a largenumber of samples is multiplexed on single lane, sample preparation canbe expected to dominate the cost of the assay, with sequencingaccounting for a small fraction of the overall cost. Although in thepresent work bar-codes were introduced as part of the Illumina samplepreparation pipeline, an equivalent means of distinguishing amongsamples would be to introduce bar-codes into the library of reporterplasmids. This configuration would permit sample pooling prior tosequencing library preparation, leaving the cost of cell culture andtransfection reagents as the major remaining cost of the assay.

HCR-seq constitutes a paradigm shift in the quantitation of DNA repaircapacity because of the ability to measure the repair of any lesion thatinduces transcriptional mutagenesis. This is an important advancebecause many DNA lesions can be bypassed by human RNA polymerase. As aresult, they cannot be detected by a conventional HCR assay without therequirement for reporters specifically engineered to give a functionalreporter protein in the presence of the lesion [26]. HCR-seq has no suchrequirement, so rare or unexpected transcriptional mutagenesis eventscan be detected. Base misincorporation opposite DNA lesions by RNApolymerase during transcription often mirrors that by DNA polymeraseduring replication. Thus, most mutagenic lesions have a transcriptionalmutagenic signature [27]. The HCRseq strategy should therefore be usefulin DNA repair capacity measurements for nearly any pathway. The data inFIG. 7 illustrate the power of this unbiased approach to detect rareevents that are specific to transcription of damaged DNA. Because theplasmids are not replicated in the cell, and sequence changes wereobserved at an elevated rate in repair deficient cells, these are likelyto reflect transcriptional mutagenesis events at unrepaired DNA lesionsin the transcribed strand.

In addition to the possible clinical applications described above,HCRseq allows for the elucidation of new biological phenomena in theresearch setting. The observed 6-8 base pair deletions at ApA sequencesopposite a site specific CPD are consistent with a recent report thatbulky, helix-distorting lesions can be bypassed by human RNA polymeraseII, giving rise to rare transcriptional mutagenesis events, includingdeletions [28]. However, the observation of frequent basemisincorporation opposite a CPD by RNA polymerase II appears to bewithout precedent. A recent study indicated that CPD bypass followed theso-called “A-rule”, resulting in error-free bypass [29]. In that study,base misincorporation was observed, however subsequent extension oftranscripts beyond a misincorporated base was strongly inhibited. Thepresent data provide evidence of error-prone transcriptional bypass ofbulky DNA lesions in human cells followed by completion of thetranscript. A lower limit (about 10%) for the frequency of these bypassevents can be estimated from the data in FIG. 7. Since it is expectedthat reporter plasmids that have already been repaired or did notcontain a thymine dimer at the time of transfection will be transcribedat a higher rate, and because error-free bypass cannot be distinguishedfrom transcripts arising from repaired plasmid, the rate of bypass maybe higher than 10%.

Conclusions

HT-HCR and HCRseq represent powerful new tools for high throughputmeasurements of human DNA repair capacity. HT-HCR permits the rapid andsimultaneous measurement of at least 4 independent repair processes in asingle assay. HCR-seq has the potential to measure thousands of repairprocesses in a single assay, and expands the type of lesions whoserepair can be measured to include those that do not block transcription.The use of sample-specific bar-codes permits the simultaneousmeasurement of repair capacity in multiple individuals, therebyminimizing interexperimental variability. The use of barcodes withHCR-seq also has the potential to reduce the cost and labor required forDNA repair capacity measurements to a level compatible with large scaleepidemiological studies and clinical diagnostic/prognostic applications.Both assays hold an advantage over methods requiring cell lysis becausethe intact DNA repair machinery of a living cell acts on nuclear DNA,thus increasing the likelihood of recapitulating physiological DNArepair phenotypes. As a research tool, the unbiased HCRseq approach alsohas the potential to reveal previously unknown mechanisms of DNA repairand damage tolerance.

TABLE 1 Cell lines used in this study. To facilitate comparison of data,the seven individuals from whom both lymphoblastoid and fibroblastcultures were derived have been assigned indexes α through η. Cell LineIndividual Cell Type Genotype NER Phenotype GM01630 (α) PhoebeFibroblast XPA Severe GM01953 Rhea Lymphoblastoid WT None GM02246 CronusLymphoblastoid XPC Moderate GM02249 Mnemosyne Lymphoblastoid XPC MildGM02253 Oceanus Lymphoblastoid XPD Severe GM02344 (α) PhoebeLymphoblastoid XPA Severe GM02345 Coeus Lymphoblastoid XPA SevereGM03657 (β) Hyperion Lymphoblastoid WT None GM03658 (β) HyperionFibroblast WT None GM07752 (γ) Tethys Lymphoblastoid WT None GM07753 (γ)Tethys Fibroblast WT None GM14878 (δ) Theia Lymphoblastoid XPC Very MildGM14879 (δ) Theia Fibroblast XPC Very Mild GM21071 (ε) Themis FibroblastXPB Severe GM21148 (ε) Themis Lymphoblastoid XPB Severe GM21677 (ζ)Crius Lymphoblastoid WT None GM21833 (η) Iapetus Lymphoblastoid WT NoneGM23249 (ζ) Crius Fibroblast WT None GM23251 (η) Iapetus Fibroblast WTNone

TABLE 2 Combinations of reporter plasmids used in each experiment. Doseto plasmid is given in units of J/m². Combination BFP Cyan GFP mOrangemPlum #1 600 0 800 200 400 #2 0 200 400 600 800 #3 0 200 T< >T¹ 400 800¹Site specific thymine dimer

TABLE 38 bar-coded samples submitted for deep sequencing on a single lane.Number of Reads Damage to Sequencing aligned to Sample Cell line PlasmidBarcode Type reporters A GM2344 No CTACTG RNA   501791 B GM2344 YesGGCAAC RNA   141195 C GM1953 No TCGTCA RNA   503521 D GM1953 Yes TAGGCTRNA   422704 E GM2344 No ATGATA DNA 14083169 F GM2344 Yes CAAGTT DNA19129464 G GM1953 No GTCCAG DNA 22275922 H GM1953 Yes TGGACC DNA22511231

TABLE S1 Cufflinks and Tophat parameters. Value Description Tophatparameter min-anchor-length 6 TopHat will report junctions spanned byreads with at least this many bases on each side of the junction. Notethat individual spliced alignments may span a junction with fewer thanthis many bases on one side. However, every junction involved in splicedalignments is supported by at least one read with this many bases oneach side. This must be at least 3 and the default is 8.splice-mismatches 0 The maximum number of mismatches that may appear inthe “anchor” region of a spliced alignment. The default is 0.min-intron-length 10 minimum intron size allowed in genomemax-intron-length 1000000 maximum intron size allowed in genomemin-isoform-fraction 0.0 The minimum frequency of any isoform toconsider. The default is 0.15 max-multihits 20 Instructs TopHat to allowup to this many alignments to the reference for a given read, andsuppresses all alignments for reads with more than this many alignments.The default is 20 for read mapping. no-novel-juncs True Only look forreads across junctions indicated in the supplied GFF or junctions file.segment-length 20 Each read is cut up into segments, each at least thislong. These segments are mapped independently. The default is 25.library-type fr- library prep used for input reads unstrandedsolexa1.3-quals True As of the Illumina GA pipeline version 1.3, qualityscores are encoded in Phred-scaled base-64. Use this option for FASTQfiles from pipeline 1.3 or later. Cufflinks parameters min-intron-length10 minimum intron size allowed in genome max-intron-length 1000000maximum intron size allowed in genome min-isoform-fraction 0.0 Theminimum frequency of any isoform to consider. The default is 0.15library-type fr- library prep used for input reads unstrandedcompatible-hits-norm True count hits compatible with reference RNAs onlymulti-read-correct True use ‘rescue method’ for multi-reads (moreaccurate) frag-bias-correct True use bias correction - reference fastarequired

TABLE S2 Read counts for RNA-seq samples and numbers of aligned readsusing Tophat. XPA mut undam XPA mut dam Norm undam Norm dam Total reads22903329 21345212 23454955 24408453 Unaligned 428195 358787 448506453861 Aligned to all Total 26504278 24805211 27527409 28496167Ambiguous 4495789 4250691 4992157 5018134 Unique 22008489 2055452022535252 23478033 Aligned to human Total 26002487 24664016 2702388828073463 genome Ambiguous 4430397 4246605 4926437 4975358 Unique21572090 20417411 22097451 23098105 Aligned to reporter Total 501791141195 503521 422704 Ambiguous 126142 7612 124807 81196 Unique Total375649 133583 378714 341508 BFP 28462 19299 28104 26471 AmCyan 14370090582 155397 184217 GFP_615 39654 12250 38082 25282 mOrange 49208 524946614 38222 mPlum 114625 6203 110517 67316

TABLE S3 RPKM values for the five reporter genes across samples.Reporter RPKM values gene XPA mut undam XPA mut dam Norm undam Norm damBFP 2480.96 1792.89 2444.02 2254.98 AmCyan 14427.1 9816.38 15365.917441.2 GFP_615 3281.55 1077.19 3148.35 2057.57 mOrange 6642.74 683.9946356.26 5046.36 mPlum 14995 829.241 14584.1 8869.09

TABLE S4 Read counts for RNA-seq samples and numbers of aligned readsusing TopHat. XPA mut undam XPA mut dam Norm undam Norm dam Total reads15477805 22428323 24517460 25680796 Unaligned 2541912 3488151 34615623618298 Aligned to all Total 14097831 19153649 22299508 22546451Ambiguous 1416044 643871 1663555 872727 Unique 12681787 1850977820635953 21673724 Aligned to human Total 14662 24185 23586 35220 genomeAmbiguous 2396 3755 3676 5589 Unique 12266 20430 19910 29631 Aligned toreporter Total 14083169 19129464 22275922 22511231 Ambiguous 1413648640116 1659879 867138 Unique Total 12669521 18489348 20616043 21644093BFP 1266523 6137156 2162883 3159934 AmCyan 3571988 6115952 1036018314648713 GFP_615 1376690 3459175 908607 287825 mOrange 2388861 17660142737414 1610236 mPlum 5479030 1651015 6106623 2804324

TABLE S5 RPKM values for five reporter genes generated by TopHat andCufflinks analysis of the DNA sequence data. Reporter genes XPA mutundam XPA mut dam Norm undam Norm dam BFP 142139 501211 127941 212433AmCyan 541422 586301 862136 1103440 GFP_615 138125 273507 46110.213027.7 mOrange 154654 118996 116079 102183 mPlum 587504 114037 406956244267

TABLE S6 Genes with log2 fold change >= 1 when comparing cellstransfected with undamaged plasmid to those transfected with damagedplasmids. RPKM cutoff Sample Gene Name Chr Bp Log2 Fold Change RPKM >= 5XPA mut RPL21 chr13 27825691-27830702 1.480810166 DDX39B chr6_apd_hap12812683-2821649 2.297351567 GFP_615 GFP_615  0-951 −1.607104673 SMN2chr5 69345349-69373422 −1.265076988 SMN2 chr5 70220767-70248842−1.282415323 mOrange mOrange  0-942 −3.279722873 mPlum mPlum  0-912−4.176546263 Normal ARRDC3 chr5 90664540-90679149 1.420496975 SCARNA27chr6 8086640-8086766 1.547032054 RPKM >= 1 XPA mut OSCP1 chrl36883506-36916086 1.052446063 RPL21 chr13 27825691-27830702 1.480810166SELM chr22 31500762-31503551 1.269204139 LOC100505894 chr587564698-87732491 1.183911485 HIST1H2BH chr6 26251878-262523031.097113886 HIST1H2BN chr6 27806439-27806888 1.292384444 DDX39Bchr6_apd_hap1 2812683-2821649 2.297351567 GFP_615 GFP_615  0-951−1.607104673 HIST2H3A chr1 149812258-149812765 −1.009472345 HIST2H3Achr1 149824180-149824687 −1.009472345 FAM45B chr10 120863628-120897376−1.415998178 PLSCR3 chr17 7293046-7298162 −1.085829966 SMN2 chr569345349-69373422 −1.265076988 SMN2 chr5 70220767-70248842 −1.282415323LEAP2 chr5 132209357-132210582 −1.210275283 HIST1H2BO chr627861202-27861669 −1.294428563 mOrange mOrange  0-942 −3.279722873 mPlummPlum  0-912 −4.176546263 Normal C1orf162 chr1 112016603-1120211341.023536205 ZNF826P chr19 20578625-20607771 1.359917941 IL29 chr1939786964-39789312 1.01622561 C21orf119 chr21 33765441-337662661.058253039 ARRDC3 chr5 90664540-90679149 1.420496975 SPATA24 chr5138732455-138739776 1.170374309 EEF1E1-MUTED chr6 8013799-81028281.336028163 SCARNA27 chr6 8086640-8086766 1.547032054 RPPH1 chr1420811229-20811570 −1.018946998 PLSCR3 chr17 7293046-7298162 −2.245649866TMEM238 chr19 55890611-55895627 −1.080267671 EIF4EBP3 chr5139927250-139929163 −1.563613927 GTF2IP1 chr7 72569025-72621336−1.031276973 RMRP chr9 35657747-35658015 −1.351587952

TABLE S7 Observed frequency of sequence changes in reporter transcriptsat positions corresponding to the site specific CPD. GM01953 GM02344Undamaged Damaged Undamaged Damaged Delete ApA opp 0.00% 0.29% 0.00%1.07% T< >T AA Del, Randomly 17.9 23.7 14.0 42.5 Dam¹ 5′A-->G 0.07%1.87% 0.15% 8.97% 3′A-->T 0.00% 0.34% 0.00% 1.89% ¹Deletions per millionmapped reads

TABLE S8 Observed frequency of sequence changes in reporter transcriptsat positions corresponding to the site specific CPD (DNAseq data).GM01953 GM02344 Undamaged Damaged Undamaged Damaged Delete ApA opp 0.00%0.09% 0.00% 0.44% T< >T AA Del, Randomly 1.4 9.5 0.0 26.8 Dam¹ 5′A-->G0.07% 1.75% 0.07% 9.73% 3′A-->T 0.01% 0.17% 0.01% 1.21% ¹Deletions permillion mapped reads

Example 2: Construction of Reporter Constructs

Most of the multiple cloning (MCS) of plasmid pmax Cloning was excisedto minimize the size of the reporter, leaving only the KpnI and SacIrestriction sites (see MCS diagram at bottom of FIG. 15), plus tworestriction sites (illustrated in blue) introduced for convenientsubcloning of reporter genes of the following general structure:KpnI-NheI-Reporter_Gene-HindIII-Sacl. The resulting reporter plasmidslack any mammalian origin of replication, and express no other geneexcept for the reporter in mammalian cells. This construction was usedto characterize DNA repair mechanisms independently DNA replication.However, plasmids that can be replicated in human cells are used tostudy the repair and tolerance of DNA damage in the context of DNAreplication.

Example 3: Multicolor Fluorescent Reporter Strategy

Damage induced in DNA by UV-irradiation is known to block transcription.As this damage is repaired, transcription is restored. As shown in FIG.16, 5 five plasmids encoding separate reporter proteins, BFP (blue),AmCyan (Cyan), GFP (Green), mOrange (Orange) and mPlum (Red) areco-transfected into cells to establish a control level of fluorescentreporter expression from undamaged plasmids, typically at 24 hours (topof FIG. 16). 4 Four reporters are selected as repair capacity reporters,and normalized to a fifth color (in this case BFP), that is included asa transfection control. To measure nucleotide excision repair capacity,a different dose of UVC irradiation ranging from 0-800 J/m2 is deliveredto each reporter (in general, either the dose or the type of damage canbe varied, depending on the DNA repair pathway to be interrogated). Asdescribed, BFP is left undamaged, and its expression level used as atransfection control. The damaged reporters are mixed and co-transfectedinto cells. After allowing for a period of DNA repair, the level ofexpression is determined for each reporter and expressed as a percentageof control (undamaged) reporter expression. Expected data are shown inFIG. 16, right panels.

Example 4: Flow Cytometric Detection and Measurement of ReporterFluorescence

As shown in FIG. 17, BFP and AmCyan are excited using a 405 nm laser,and detected in the pacific blue and AmCyan detectors, respectively. GFPis excited at 488 nm and measured in the FITC detector. mOrange andmPlum are excited at 561 nm and detected in the PE and PE-Cy5-5detectors, respectively. TOPRO®-3, used to exclude dead cells from theanalysis, is excited at 634 nm and measured in the APC detector. Usingpositive and negative controls, a positive region defined as follows isestablished for each reporter: Cells expressing the reporter fall inthis region, and when the reporter is excluded from a transfection inwhich all other reporters are present, no cells fall in this region.Expression for each reporter is calculated as the percentage of cellsappearing in the positive region, multiplied by their mean fluorescenceintensity (MFI+) in the appropriate detector.

Example 5: Dose Response Curve Corresponding to the MulticolorFluorescent Reporter Strategy

Seven cell lines previously characterized for their nucleotide excisionrepair capacity (Athus et al. Cancer Res 1991 51, pp. 5786-5793) werestudied using the developed assay. Expression of fluorescent reporterprotein from damaged plasmids (AmCyan, 200 J/m2, GFP, 400 J/m2; mOrange600 J/m2; mPlum, 800 J/m2) was plotted in FIG. 18 as a percentage ofexpression from the respective undamaged control plasmids. A linecorresponding to 37% was drawn in grey; this corresponds to the dose atwhich there is, on average, one transcription blocking event perreporter. The dose at which the curves shown in FIG. 18 cross this lineis defined as D_(o). This quantity represents a numerical measure ofrepair capacity with higher numbers indicating a higher repair capacity.

Example 6: Sequencing Based Detection of Reporter Transcripts

A library of plasmids containing short DNA barcodes (BC1, BC2, . . .BCn) within the transcribed region of a reporter gene under the CMVpromoter is generated (FIG. 20). Cells are transfected with the libraryof undamaged, bar-coded plasmids to establish a control level ofreporter expression. Transcripts are counted using deep sequencing asseen in FIG. 21. A unique dose or type of DNA damage is introduced intoeach of the bar-coded plasmids. As with the flow cytometric systemdescribed in FIG. 16, one bar-coded reporter (designated in FIG. 20 asBC1) is left undamaged for use as a transfection control. The mixture ofdamaged plasmids is co-transfected into cells, and after allowing timefor DNA repair, transcripts are isolated from the cells, counted, andreported as a percentage of expression from the undamaged control.

Example 7: Methodology for Analysis of Reporter Transcripts by NextGeneration Sequencing

Cells are harvested, lysed, and their total RNA purified, and thenfurther purified to mRNA using commercially available kits. Reversetranscription of reporter mRNAs to their corresponding cDNAs is achievedwith reverse transcriptase, using either a poly-dT oligonucleotide, asshown in FIG. 21, or a reporter-specific oligonucleotide as a primer.Signal to noise is increased by subsequently PCR amplifying the reportercDNAs with primers specific to the 5′ and 3′ UTR of the reportertranscripts (see FIG. 15 and FIG. 22). cDNA is then either cloned intoplasmid vectors for conventional sequencing or submitted for nextgeneration sequencing. In the case of next generation sequencing,additional barcodes may be appended at this stage to the 5′ and 3′ endsof cDNAs from replicate transfections, multiple time points or separatesamples to enable multiplex sequencing in a single lane. Total reporterexpression is measured as the number of sequencing reads that can beunambiguously assigned to the identifying bar-coded region of thereporter. Depending on the type of DNA damage, sequencing reads may beanalyzed more deeply as shown FIG. 23. The primers used to amplify cDNAmay be placed so as to flank any specific region of interest in thegene, as is needed in the case of site-specific DNA damage. (FIG. 21)

Example 8: Gel Purification of cDNA Amplified Using Reporter-SpecificPrimers

In this experiment, the RFP plasmid was either left undamaged or exposedto 800 j/m2 and then co-transfected with EGFP control plasmid intolymphoblastoid cells (FIG. 22). Total RNA was extracted, poly-dTpurified, and reverse transcribed with poly-dT primers as described inFIG. 21. The lanes were used as follows: Lanes: 1—Water Control, Lane2/3—Plasmid Positive Controls, 4/5—cDNA from undamaged HCR, +/− reversetranscription (RT), 6/7—cDNA from 800 j/m2 HCR, +/−. As the UTR primersamplify a region spanning the 136 bp chimeric intron in the pmax vector,plasmid templates were expected to give a slightly longer amplicon.Higher resolution experiments may be used to provide even strongerconfirmation for the mass differences seen in the gel. The cDNAgenerated and amplified by these methods was cloned into plasmids andsequenced (see Table 2).

Example 9: Evaluating the Utility of Single Nucleotide ResolutionTranscript Analysis

The fidelity of transcription in the presence of a given type of DNAdamage may be evaluated from the frequency of errors detected intranscripts associated with a given bar code. In this example, thelesion O⁶-methylguanine as indicated in red in FIG. 23, is sitespecifically incorporated into a reporter plasmid, 9 nucleotidesdownstream from a 9-nucleotide bar code (shown in grey). This lesionresults from the miss-incorporation of uracil in mRNA with a frequencyof approximately 25% (Dimitri, A., et al., Nucleic Acids Research, 2008.36(20): p. 6459-6471.) Expected data are shown in the column graphs.During cDNA synthesis, the incorrect uracil is reverse transcribed to T,leading to a mixed population of cDNAs associated with that bar code.This is illustrated in FIG. 23 in the sequences shown with percentagesimmediately below the diagram of the reporter construct. Repair capacityis estimated from the rate at which the fraction of transcriptscontaining the correct nucleotide at this position increases. A timecourse is then carried out, ranging from 0 to up to 96 hours. Thefraction of transcripts containing the correct nucleotide at theposition of interest is calculated at each time point. Relativeabundance of repaired to unrepaired substrate is at least equal to theratio of correct transcripts to incorrect transcripts. The estimatedtime-dependent ratio of repaired to unrepaired substrate is used tocalculate a relative rate of repair that can be compared among celllines. This method may be generalized to any DNA lesion that alters thesequence transcribed from the reporter. In this example, where there isno inhibition of transcription, 100% total transcription of the damagedreporter is shown. However, some lesions may be expected to alter boththe extent and fidelity of transcription. Both phenomena may be measuredsimultaneously by this method without modification.

Example 10: Introduction of Site-Specific DNA Lesions into ReporterPlasmids

Single strand nicking sites were introduced flanking the position atwhich the lesion was to be introduced. The plasmid was cut with thenicking enzyme, and the excised strand was displaced by a singlestranded oligonucleotide, shown red in FIG. 24, that is complimentary tothe single stranded region created in the double-nicked plasmid. Thedisplacing oligonucleotide contained a lesion, indicated in FIG. 24 as ared arch, at the desired position. Following annealing, theoligonucleotide was ligated into the plasmid.

Example 11: Introduction of a Site-Specific Thymine Dimer into the PmaxReporter Plasmid

Two nicking sites for the Nb.Bpu101 nicking endonuclease were introducedinto the pmax GFP reporter. Following cutting, the plasmid was incubatedwith a large excess of modified oligonucleotides complimentary to thesequence spanned by the nicking sites in the plasmid. The modifiedoligonucleotides contained a site-specific thymine dimer, indicated as astar (*) in FIG. 25. These oligonucleotides displaced the excised nativeoligonucleotide. Following polynucleotide kinase treatment, theoligonucleotide was ligated into the plasmid. Successful ligation andfunctional characterization of the resulting reporter are illustrated inFIG. 26.

Example 12: Verification of a Site-Specific DNA Damage ContainingReporter

Verification of a site specific DNA damage containing reporter wasverified by gel electrophoresis. These results can be found in FIG. 26:Uncut reporter plasmid in lane #1 ran at approximately 2 kb. The nickedplasmid in lane #2 ran at close to 4 kb. Ligase failed to yield a closedcircular product between the displacing thymine dimer containing oligo(T< >T) and the nicked reporter in the absence of PNK (lane 3). However,in the presence of PNK, the T< >T oligo is successfully ligated into thereporter plasmid (lane #4, 2 kb band). This product can be cut with T4PDG, a bifunctional glycosylase that creates a single strand break onlywhere pyrimidine dimers are present in DNA. The exclusively nicked DNAin lane 5 confirms that the closed circular product in lane #4 containsthe pyrimidine dimer. Randomly induced UV damage also makes the reporterplasmid a substrate for T4 PDG (Lane #6). On the right, we see thatReporter expression is partially blocked in the reporter constructcorresponding to lane #4 in the gel. As expected, the XPA cell line,which is deficient in the Nucleotide Excision Repair pathway that actson thymine dimers, shows more severely reduced fluorescent reporterexpression of the site-specific reporter, relative to the undamagedcontrol. We are preparing to make more substrates like this one, withdifferent types of damage, for use in a high throughput sequencing basedHCR, as described above in FIGS. 20, 21 and 23.

Example 13: Four Basic Reporter Constructions Comprise the Library ofReporters to be Used with Next Generation Sequencing

FIG. 27 shows four basic reporter constructions that comprise thelibrary of reporters to be used with next generation sequencing. Allreporters have a mammalian promoter, which could be the CMV promoterwhere very high expression is desired, or an inducible/repressiblepromoter that permits adjustable expression. The latter has utility instudying transcription coupled repair of DNA damage. Each reporter has aunique bar code. The bar code identifies the lesion that is present inthe reporter, and can also be used to multiplex samples (with adifferent bar code associated with each subject or cell line beingstudied). Sequence changes in transcripts in the sequence spaceindicated as “lesion” report on transcriptional mutagenesis. In Example1, the entire fluorescent reporter sequence was used as the bar code,and as the sequence space that reported on transcriptional mutagenesis.All reporters contain a lesion, and have in common 5′ and 3′ PCRamplification sequences (5′Amp and 3′Amp) that are located in such amanner that the resulting amplicon spans both the lesion and the barcode. The lesion may be located 3′ (A) or 5′ (B) to the bar code. Amammalian origin of replication may be absent (A and B), or present. Thelatter is useful in studying the repair of lesions in the context of DNAreplication.

Example 14: Gating Procedure for 6-Color Flow Cytometric Detection of 5Fluorescent Reporters and One Dead Cell Stain

FIG. 28 shows a primary gating scheme for TK6 lymphoblastoid cells. Thepopulation hierarchy is shown at the bottom of the figure; A mainpopulation P1 was established in the SSC-A vs FSC-A plot. Nested withinP1, a secondary population P2 was established using the FSC-H vs. FSC-Wplot. Further nested within this population, a tertiary population P3was established. P2 and P3 were used to exclude doublets and higherorder aggregates of cells. Finally, the cells in P3 were separated intotwo populations, live and dead. Cells were incubated at room temperaturefor 5 minutes in 100 nM TOPRO-3. The stain was left in the cellsuspension, and fluorescence was measured in the APC channel (Excitationwith a 634 nm laser). Dead cells in the higher staining population wereunable to exclude the dye. Live cells were analyzed further for theirfluorescence in the other five colors, namely the fluorescent reporterproteins tagBFP, AmCyan, EGFP, mOrange, and mPlum. The procedure foridentifying cells positive for each of these reporters is given below.

FIG. 29 shows Negative controls (mock transfected cells). Cells thathave been subjected to transfection conditions in the absence ofexogenous plasmid DNA are used to establish regions in each channelcorresponding to cells that are not expressing fluorescent reporters.Gates are drawn so as to exclude at least 99.9% of the untransfectedcells. Gates needed to distinguish positive from negative cells arecircumscribed in FIG. 29 with boxes in colors that correspond to thecolor of the respective reporter indicated on the X-axis of each plot(BFP, blue, Pacific blue detector; AmCyan, cyan, AmCyan Detector;mOrange, orange, PE detector; mPlum, red, PE-Cy5-5 detector; GFP, green,FITC detector).

FIG. 30 shows an example of a single color control. Cells appearingsimultaneously in both P13 and P14 are defined to be positive forAmCyan. (An example showing why multiple gates are needed is seen belowfor GFP). Compensation has been adjusted so that, as nearly as possible,cells positive for AmCyan have the same MFI for each of the otherdetectors as do cells negative for AmCyan. Similarly, cellssimultaneously in P9 and P11 are positive for BFP, and Cells appearingin all three gates, P15, P16, and P18 are positive for GFP. 3 gates areneeded to define GFP positive cells because of spectral overlap. This isseen in gates P15 and P16, where cells positive for AmCyan also appearto be positive for GFP, despite spectral compensation. However, gate P18excludes the cells responsible for the false positives in P15 and P16.Using this system of gates, when GFP is excluded from a transfection,99.9% or more of cells are detected as negative for GFP, regardless ofthe presence of other reporters (not shown). For the reporters describedhere, 9 gates were the minimum required to establish regions such thatat least 99.9% of cells not transfected with a given reporter areexcluded from the positive population. A general approach is describedbelow.

Compensation is applied to data as follows: Cells are transfected witheach reporter of interest one at a time (single color controls). Twopopulations, positive and negative, are established for a givenreporter. Compensation in that detector is adjusted until the meanfluorescence intensity (MFI) measured for each other detector is thesame for both positive and negative populations, as defined above. Inother words, the MFI in one detector is independent of whether the cellsare positive or negative for a second detector.

The general approach to establishing a gating scheme for any set offluorescent reporters is as follows: Set compensation using single colorcontrols, as described above. To determine the positive region for agiven reporter, a plot the reporter of interest (on the horizontalaxis), against each of the other reporters (on the vertical axis) thatwere found to have significant spectral overlap with the reporter ofinterest. For each single color control, examine the plot of that coloragainst the reporter of interest. Establish a region that excludes falsepositives in the reporter channel of interest due to reporter expressionin the single color control channel. Establish a gate that takes theunion “AND” of these gates as the region corresponding to cells positivefor the reporter of interest. Repeat this process for each reporter.Then examine the “minus-one” controls, where one reporter is excluded ata time (and all others are present, for example minus Cyan in FIG. 31).Examine the minus-one transfection for the detector of interest, andagain adjust all gates to ensure that at least 99.9% of cells areexcluded from the positive “union” gate for the detector of interest.Repeat this process for the remaining reporters.

Example 15: Cisplatin and Reporter Expression

The data provided in FIG. 32 show that treatment of DNA repair reportervectors with Cisplatin suppresses florescent reporter expression in adose dependent manner. As expected the effect was even more pronouncedin cells deficient in nucleotide excision repair and DNA crosslinkrepair (XPA and XPF).

Example 16: Synthesis of a Substrate with a Site-SpecificO⁶-Methylguanine (O⁶-MeG)

FIG. 33 shows a summary of the method for the synthesis of asite-specific O⁶-MeG using single stranded closed circular DNA and anoligonucleotide containing an O⁶-MeG residue at a defined position.

1) Prepare mPlum ssDNA (+) strand as described by digest with a nickingendonuclease specific for the (−) strand followed by digest with ExoIII,which removes the (−) strand.

2) Combine single stranded DNA with 4-fold molar excess of 30 nt oligocontaining 06-Methylguanine

3) Treat with polynucleotide kinase, heat to 85° C., slow cool to anneal

4) Add dNTPs, T4 DNA polymerase, T4 DNA ligase

5) Incubate 1 hour at 37° C.

(Baerenfaller et al. (2006) Method Enzymol 18, p 285)

Example 17: HCR Assay Using a Substrate with a Site-Specific O⁶-MeG

FIG. 34 shows an HCR assay using a substrate with a site-specificO⁶-MeG. The data show an inverse relationship between % reporterexpression using the disclosed HCR-assay and MGMT (methyl guanine methyltransferase) activity as measured by an independent method in lysates.As O⁶-MeG is repaired, transcriptional mutagenesis decreases, resultingin less expression of the wild type mPlum reporter protein.

Example 18: Comparing 2-Color Versus 5-Color Fluorescent Reporter HCR ofUV-Irradiated Plasmids

FIG. 35 provides a graph comparing 2-color versus 5-color fluorescentreporter HCR of UV-irradiated plasmids. UV HCR: XPA-deficient cell lineat 16 hours. In the 2-color experiment, 5 transfections using theplasmids pmax:GFP and pmax:mCherry were necessary:

1. pmax:GFP plus pmax:mCherry at 0 J/m².

2. pmax:GFP plus pmax:mCherry at 200 J/m².

3. pmax:GFP plus pmax:mCherry at 400 J/m².

4. pmax:GFP plus pmax:mCherry at 800 J/m².

5. pmax:GFP plus pmax:mCherry at 1200 J/m².

In the 5-color experiment, only two transfections were necessary:

-   -   1. pmax:BFP+pmax:AmCyan+pmax:GFP+pmax:mOrange+pmax:mPlum, all at        0 J/m².    -   2. pmax:BFP at 0 J/m²+pmax:AmCyan at 200 J/m²+pmax:GFP at 400        J/m²+pmax:mOrange at 800 J/m²+pmax:mPlum at 1200 J/m².

Example 19: Estimating Recombination Frequency

FIG. 36 provides an illustration showing a method of estimation ofrecombination frequency. In one transfection (top) cells areco-transfected with 1 microgram of pmax:mCherry as to control fortransfection efficiency, plus 0.5 micrograms of a 5′-truncated(nonfluorescent) linearized GFP reporter plasmid pD5GFP (see KiziltepeT. et al, Chemistry & Biology, 2005. 12(3): p. 357-369), plus 5micrograms of a 3′truncated GFP reporter plasmid. If 100% of thelinearized plasmid is repaired by homologous recombination, the expectedfluorescence signal is equal to that obtained from a separateco-transfection (bottom) with 0.5 micrograms of full length GFP reporterplasmid plus 1 microgram of pmax:mCherry reporter plasmid.

Example 20: Double Strand Break Induced Recombination

FIG. 37 provides a graph showing a 25-fold range of HR repair capacityover several cell lines in DSB “induced” recombination. As expected, areduced recombination frequency is observed in a cell line deficient forBRCA1.

Example 21: Mismatch Repair Substrate

FIG. 38 provides an illustration showing a mismatch repair substrate(See also Zhou, B. S. et al, Anal. Biochem. 388, 167-169, (2009))wherein the sequence in the transcribed strand of the reporter plasmidencodes a non-fluorescent plasmid. Restoration of the wild type sequencein the transcribed strand can result from mismatch repair activity,leading to expression of the wild type (fluorescent) reporter proteinmOrange.

Example 22: Multiple Lesions in a Single Plasmid

FIG. 39 provides an illustration showing multiple lesions in a singleplasmid. A wild type closed circular single stranded DNA comprising thenon-transcribed strand of the pmax:mOrange fluorescent reporter plasmidhas been annealed to the complementary (transcribed) strand of a plasmidwith three mutations, each of which results in a non-fluorescentreporter protein. A single base insertion at position 50 results in asingle base loop. Base substitutions at positions 215 and 299 result inA:C and G:G mismatches, respectively. All combinations of the mutationsalso yield reporter plasmids that express non-fluorescent reporterproteins. Only repair of all three lesions results in fluorescence. Thisreduces background that may arise from other repair mechanisms (such asBER) that may act locally on some mismatches and loops; the length ofmismatch repair tracts is sufficient to repair all three lesions in asingle repair event.

Example 23: Mismatch Repair and Multiple Lesions

FIG. 40 provides a graph showing that the differential between mismatchrepair proficient (MMR+) and deficient (MMR−) improves with multiplelesions. HCT116 cells are (MMR−) because they are deficient for themismatch repair protein MLH1. HCT116+3 cells are complemented with MLH1expressed from the human chromosome #3, and are therefore MMR+.

Example 24: Alkylation Damage Repair and Transcription Inhibition

FIG. 41 provides a graph showing that the inhibition of transcription isnot detected when a fluorescent reporter plasmid is treated with thealkylating agent MNNG and reporter expression is assayed by flowcytometry 16 hours after transfection. In this example a plasmid wastreated for 4 hours in 0.8 mM MNNG (Cell lines #4-22 are Corriell celllines from apparently healthy individuals). Treatment with MNNG inducesseveral types of alkylation damage in DNA, including O⁶-MeG. TK6 cellsare deficient for MGMT, the enzyme that repairs O⁶-MeG by directreversal, and TK6+MGMT has been complemented with the enzyme to restorerepair capacity. O⁶-benzylguanine (BnG) inactivates MGMT, renderingcells treated with the compound unable to repair O⁶-MeG (TK6+MGMT+BnG).The lack of significant differences in the extent of reporter expressionin these cell lines indicates ordinary host cell reactivation assays arenot amenable to measurements of O⁶-MeG repair capacity.

Example 25: Non Fluorescent mPlum Variant

FIG. 42 shows that a point mutation (T208C) results in a non-fluorescentmPlum variant S70P. Of 500,000 cells analyzed—virtually no plum positivewas found.

Example 26: Mutations Induced in mPlum Variant

FIG. 43 is an illustration showing that when O⁶MeG is present in thetranscribed strand, some mRNA will contain U producing wild type mPlumprotein. The mutation T208C results in a guanine at the correspondingposition in the transcribed strand. Transcripts therefore contain thecodon CCC (proline) instead of the wild type UCC (serine). However,since O⁶MeG is transcribed as “U” instead of “C” with approximately 25%frequency, some transcripts produced from plasmids containing thislesion will have the wild type sequence, and will be translated intofluorescent protein.

Example 27: Assay for O⁶Methylguanine HCR

FIG. 44 is an illustration providing an assay for O⁶Methylguanine HCR.An increase in fluorescence is expected when the reporter plasmidcontaining the mPlum:T208C mutation is treated with an agent thatinduces O⁶MeG. Cells that are able to repair the lesion will exhibitreduced fluorescence.

Example 28: MNNG Induced Plasmid Induces Plum Positive Variants

FIG. 45 shows the results for TK6, which are MGMT-deficient. 500,000cells were analyzed at 16 hours post-transfection—only a few hundredplum positive with MNNG treated plasmid because random alkylationproduces O⁶MeG at a low frequency. The method of introducing asite-specific O⁶MeG (Example 17) is much more efficient and so requiresfewer cells.

Example 29: MGMT and Reporter Signal

FIG. 46 is a bar graph showing that the lack of signal is MGMTdependent. Reporter expression has been normalized to that for TK6. Asexpected, the highest reporter expression is observed in MGMT deficientcells. Cells complemented with MGMT (TK6+MGMT) exhibit at least 25-foldlower reporter expression. As further confirmation that reporterexpression is MGMT- and O⁶MeG-dependent, cells incubated withO⁶Benzylguanine before and after transfection (TK6+MGMT+BnG) showelevated reporter expression similar to that of the MGMT deficient TK6cells.

Example 30: Comparison of MGMT Activity

FIG. 47 shows a preliminary comparison of reporter expression frommPlum:T208C reporter plasmids randomly alkylated with MNNG withindependent characterization of MGMT activity in extracts. (Fry et alGenes. Dev. 2008 (22) p 2621). As expected, and similar to what wasobserved when cells were assayed using a plasmid with asite-specifically engineered O⁶MeG lesion (See Example 17), an inverserelationship is observed between the extent of reporter expression andthe amount of MGMT activity found in cell lysates.

Example 31: Measurement of NER and HR in Single Assay

FIG. 48 shows that measurement of NER and HR in a single assay yieldsthe same information as separate measurements. This experimentdemonstrates the ability to measure DNA repair capacity in multiplerepair pathways using a single assay and methodology. Multiplefluorescent reporters with different colors corresponding to differentrepair pathways (for example the mOrange reporter for mismatch repair inExample 23 and the mPlum reporter for direct reversal of alkylationdamage in Examples 17 and 30) can be combined in a modular,interchangeable format to assay cells for global DNA repair capacity.

Example 32: HCR and Etheno (ϵ) Lesions

FIG. 49 shows HCR of plasmids containing etheno (ϵ) lesions. Massspectrometric quantitation confirms concentration-dependent induction ofetheno adducts in plasmid DNA by chloroacetaldehyde (CAA). Adose-dependent decrease in fluorescent reporter expression is observed,with significant differences between lymphoblastoid cell lines derivedfrom two individuals with no known mutations in the pathways known torepair DNA base etheno adducts, suggesting possible inter-individualvariability in repair capacity for DNA lesions induced by CAA.

Example 33: Etheno (ϵ) Lesions and Base Excision Repair

FIG. 50 shows that mouse cells deficient in BER and direct reversal for(ϵ) lesions repair (Aag, Alkbh2, Alkbh3 null) exhibit reduced reporterexpression from plasmids damaged with CAA, suggesting that some lesionsrepaired by these proteins at least partially block transcription.

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The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by examples provided, since theexamples are intended as a single illustration of one aspect of theinvention and other functionally equivalent embodiments are within thescope of the invention. Various modifications of the invention inaddition to those shown and described herein will become apparent tothose skilled in the art from the foregoing description and fall withinthe scope of the appended claims. The advantages and objects of theinvention are not necessarily encompassed by each embodiment of theinvention.

The contents of all references, patents and published patentapplications cited throughout this application are incorporated hereinby reference in their entirety, particularly for the use or subjectmatter referenced herein.

What is claimed is:
 1. A method of determining DNA repair capacity inmultiple pathways simultaneously in a cell, the method comprising:introducing multiple DNA repair reporter vectors into a cell, whereinthe cell has been isolated from a subject, wherein each of the multipleDNA repair reporter vectors comprises a distinct DNA lesion for eachpathway and a reporter sequence encoding a distinct fluorescent reporterprotein, exposing the cell to conditions to allow transcription ofreporter mRNA and translation of the encoded distinct fluorescentreporter protein to take place within the cell and processing the cellto detect a change in a fluorescence signal for each vector anddetermining the capacity of the cell to process the multiple DNA repairreporter vectors thereby determining the DNA repair capacity in themultiple pathways simultaneously in the cell based on the presence orabsence of the fluorescent signal for each vector.
 2. The method ofclaim 1, wherein the method involves determining DNA repair capacity ofa subject.
 3. The method of claim 2, wherein the cells obtained from thesubject are blood cells.
 4. The method of claim 1, wherein the cell isfurther processed to detect a change in the transcribed sequence of themultiple DNA repair reporter vectors that causes a change influorescence signal upon DNA lesion processing.
 5. The method of claim1, wherein the cell is further processed to detect a change in theamount of transcribed sequence of the multiple DNA repair reportervectors that causes a change in fluorescence signal upon DNA lesionprocessing.
 6. The method of claim 1, wherein DNA repair is nucleotideexcision repair, homologous recombination, non-homologous end joining,microhomology mediated end joining, direct reversal, base excisionrepair, mismatch repair or interstrand crosslink repair.
 7. The methodof claim 1, wherein the distinct fluorescent reporter protein isselected from the following fluorescent proteins: green fluorescentprotein (GFP), Discosoma sp. Red fluorescent protein (DsRed), enhancedgreen fluorescent protein (EGFP), enhanced yellow fluorescent protein(EYFP), enhanced cyan fluorescent protein (ECFP), and enhanced bluefluorescent protein (EBFP).
 8. The method of claim 1, wherein thefluorescent signal is detected using flow cytometry.
 9. A method ofdetermining the propensity of a subject to respond to a cancer treatmentregimen, the method comprising: introducing multiple DNA repair reportervectors into cells obtained from a subject, wherein each of the multipleDNA repair reporter vectors comprises a distinct DNA lesion for each ofmultiple pathways and a sequence encoding a reporter protein, exposingthe cell to conditions to allow transcription of the reporter mRNA andtranslation of reporter protein to take place within the cell, whereinthe multiple DNA repair reporter vectors comprise one or more lesions ofmultiple pathways that are representative of a cancer treatment regimen,and processing the cell to detect a change in expression of the reporterprotein and determining the capacity of the cells to process themultiple DNA repair reporter vectors simultaneously thereby determiningthe propensity of the subject to respond to the cancer treatment regimenbased on the presence or absence of the reporter protein.
 10. The methodof claim 9, wherein the cells obtained from the subject are cancercells.
 11. The method of claim 10, further comprising comparing thecapacity of the cancer cells to process the multiple DNA repair reportervectors to the capacity of non-cancer cells to process the multiple DNArepair reporter vectors.
 12. The method of claim 9, wherein the lesionsthat are representative of a cancer treatment regimen compriseDNA-crosslinks.
 13. The method of claim 9, wherein the lesions that arerepresentative of a cancer treatment regimen comprise DNA lesions thatblock transcription.
 14. The method of claim 9, wherein the lesions thatare representative of a cancer treatment regimen comprise DNA lesionsthat induce transcription errors.
 15. The method of claim 9, wherein thelesions that are representative of a cancer treatment regimen compriseDNA alkylation damage.
 16. The method of claim 15, wherein the DNAalkylation damage comprises O⁶-methyl-guanine.
 17. The method of claim15, wherein the DNA alkylation damage comprises N⁷-methylguanine. 18.The method of claim 9, wherein the reporter protein is a distinctfluorescent protein and the cell is processed to detect a change in afluorescence signal.
 19. The method of claim 9, wherein the sequenceencoding the reporter protein is selected from the group consisting ofgenes encoding proteins which increase or decrease resistance orsensitivity to antibiotics, genes which encode enzymes whose activitiesare detectable, and genes which detectably affect the phenotype oftransformed or transfected cells.
 20. The method of claim 19, whereinthe genes which encode enzymes whose activities are detectable areselected from β galactosidase, chloramphenicol acetyltransferase, oralkaline phosphatase.
 21. A method of determining DNA repair capacity inmultiple pathways simultaneously in a cell, the method comprising:introducing multiple DNA repair reporter vectors into a cell, whereinthe cell has been isolated from a subject, wherein each of the multipleDNA repair reporter vectors comprises a distinct DNA lesion for eachpathway and a unique nucleic acid sequence, exposing the cell toconditions to allow transcription of the unique nucleic acid sequence toproduce a unique mRNA sequence for each of the multiple DNA repairreporter vectors and processing the cell to detect a presence or absenceof the unique mRNAs and determining the capacity of the cell to processthe multiple DNA repair reporter vectors thereby determining DNA repaircapacity in the multiple pathways simultaneously in the cell based onthe abundance of the unique mRNAs.
 22. The method of claim 21, whereinthe multiple DNA repair reporter vectors comprises at least four DNArepair reporter vectors.
 23. The method of claim 21, wherein each DNArepair reporter vector of the multiple DNA repair reporter vectorscomprises a unique DNA lesion.
 24. The method of claim 21, wherein eachDNA repair reporter vector of the multiple DNA repair reporter vectorscomprises a specific number of DNA lesions.
 25. The method of claim 21,wherein each DNA repair reporter vector of the multiple DNA repairreporter vectors comprises a number of DNA lesions corresponding to aspecific dose of damaging agent.
 26. The method of claim 21, wherein themultiple DNA repair reporter vectors comprise lesions susceptible toprocessing by nucleotide excision repair, homologous recombination,non-homologous end joining, microhomology mediated end joining, directreversal, base excision repair, mismatch repair or interstrand crosslinkrepair.
 27. The method of claim 21, wherein processing of DNA damage isdetected by a change in the amount of transcribed sequence of the uniquemRNAs.
 28. The method of claim 21, wherein processing of DNA damage isdetected by a change in the transcribed sequence of the unique mRNAs.29. The method of claim 21, wherein the DNA lesions comprise DNA lesionsthat are bypassed by RNA polymerase.
 30. The method of claim 21, whereinthe change in the transcribed sequence or the change in the amount oftranscribed sequence is detected using DNA sequencing.